فهرست مطالب محسن سقا

we aimed at presenting a simple and efficient method for isolating and characterizing the neural crest cells.

The hen’s fertilized eggs were incubated for about 35h at 38°c and 55-60% humidity until the embryos reached to stages 10-12 according to Hamburger-Hamilton developmental stage table. Then the embryos were removed from the egg’s yolk and the neural tube was isolated and cultured for 24 h in a tissue culture dish to release neural crest cell. Then after, the neural tube was removed and allowed to NCC to expand for further 5 days. Finally, the cells were collected and subjected to PCR to study their gene expression profile.

The neural tube released NCC and these cells proliferated in culture condition. They also expressed markers including Slug, Sox9 ,and Sox10 by the RT-PCR method.

The neural tube can release NCC in culture condition and these cells can proliferate in the presence of an appropriate medium.

Acute promyelocytic leukemia is a malignancy of myeloid cells that are associated with resistance to apoptosis and differentiation arrest in promyelocytes in the bone marrow. The aim of this study was to investigate the effect of Origanum vulgare aqueous extract on apoptosis induction in an acute promyelocytic leukemia cell line (HL-60).

 The viability of HL-60 cells under treatment with various doses of Origanum vulgare extract was assessed by using a cell cytotoxic assay. Then, IC50 values​​ were determined after 24, 48, and 72 hours. The expression of Caspase8, Caspase9, Bax, Bcl2, and Nrf2 genes were measured by real-time PCR assay and different stages of apoptosis and necrosis in HL-60 cells were investigated using acridine orange/ethidium bromide double staining. Finally, data were introduced into SPSS software and analyzed by one-way ANOVA.

The results showed that Origanum vulgare extract decreased HL-60 cell survival and Caspase9, Caspase8, Bax, Bcl2, and Nrf2 gene expressions were changed after 72 hours of treatment with 1/5 of IC50 of Origanum vulgare extract. Also, morphological changes in the nucleus indicated apoptosis induction in HL-60 cells.

It seems that the aqueous extract of Origanum vulgare leaves can induce apoptosis in an acute promyelocytic leukemia cell line (HL-60) under experimental conditions.

According to application of Retinoic acid in differentiation of the stem cells to different cells and its role in apoptotic of cancer cells, the selection of appropriate dose for differentiation of stem cells is important. Thus in this study the effects of Retinoic acid in different concentrations on viability stem cells to select the appropriate dose for differentiation was investigated.
In this study, bone marrow mesenchymal stem cells were affected by different concentrations of Retinoic acid. Survival of cells was investigated after 3, 10 and 15 days of culture by MTT assay. DAPI staining was used to evaluate the number of apoplectic nuclei in treated cells after 10 and 15 days.
After three days of culture, the results showed that a large number of cells are destroyed at concentrations of 10-4, 10-3 and 10-2M of Retinoic acid, while in 10-5 and 10-6 M of Retinoic acid, it is not observed many apoptosis. Amount of 10-5M Retinoic acid after 10 days showed significant apoptosis, while the concentration of 10-6 M Retinoic acid after 15 days showed significant apoptosis compared to the control group (p It looks that 10-6 M Retinoic acid is an appropriate concentration for differentiation of mesenchymal stem cells.

According to importance of bone marrow mesenchymal stem cells in production of different cell lines, transplantation of these cells are used for treatment of many different diseases during cell therapy. Viability and proliferation of these cells after transplantation are very important. Since infertility is as public health problem in men and women, the scientists attempt to produce germ cells from differentiation of stem cells. It is supposed to use these cells for treatment of different illnesses especially for men with lack of germ cells in testes in future. However, in using stem cells for cell therapy the culture medium should be designed to increase the number of cells and efficiency of transplantation and to guarantee the health of the cells in terms of DNA damage. This study designed a suitable culture medium in order to increase the number of colonies and decrease the cell injuries.
In this study mesenchymal stem cells isolated from bone marrow of mice and exposed to retinoic acid (RA) with concentration of 10-6 M and Sertoli cells condition medium. Since mesenchymal stem cells (MSCs) produce fibroblastic colonies so the number of colonies was counted every 3 days after culture (days of 2, 5, 8, 11, and 15) under inverted microscope. The staining of ethidium bromide-acridine orange was also done for determination of apoptotic nucleus in days of 10 and 15 after culture.
The results showed that the effects of retinoic acid on grow and viability of MSCs is related to the time. It seems that RA increased the proliferation of the cells and the number of colonies increased in low time but the apoptotic cells elevated with increasing the time of culture. Condition medium of Sertoli cells also increased the proliferation of bone marrow stem cells.
According to proliferative properties of condition medium, it seems that using condition medium together with RA is better than RA alone for differentiation of MSCs to germ cells.

Telomerase as an enzyme with reverse transcriptase activity has an essential role in telomere maintenance by adding a telomere repeat sequence to the 3' end of chromosome and is important for regulating of many processes in embryonic development including cell proliferation and differentiation. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) with a self-renewal capacity are cells that can differentiate into various germ layer derivatives including neural cells and cardiomyocytes, and undergo biological changes during long-term cultivation. Hence, the passage number in which the cells expanded seems to be very important for proliferating and differentiating. This study was aimed at investigating the relationship between the telomerase activity and the growth rate of (hUC-MSCs) at different passages.
This experimental study was performed in Ardabil University of Medical Sciences, Iran, from March 2014 to December 2014. The umbilical cord samples were obtained from full-term neonate hospitalized in Alavi’s Hospital in Ardabil under sterile conditions. The umbilical vessels were clear off and the small pieces of the umbilical cord were cultured in Dulbecco's modified eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS). Then, the hUC-MSCs were harvested from passage one to three to calculate the population doubling time (PDT) and extract proteins by using CHAPS lysis buffer. Finally, the telomerase activity of the cells at different passages was measured by telomeric repeat amplification protocol (TRAP) and qRT-TRAP assays.
The hUC-MSCs population doubling time at passage from 1 to 3 were calculated as the average of 54.68±1.92, 55.03±1.71 and 69.41±2.54 hours, respectively, suggesting the higher cell passage number, the more extended PDT. The threshold cycles (CTs) for the telomerase activity also showed 30.58±0.51, 27.24±0.74 and 32.13±0.75 for the cell passage from one to three, respectively, representing the significant increasing in telomerase activity at passage two compared with the other passages (P= 0.021).
Analysis of the growth curve, PDT determination and measurement of telomerase activity of the human umbilical cord-derived mesenchymal stem cells showed that the long-term cell culture can affect on the cell proliferation and the telomerase activity.

Aim & whole-mount in situ hybridization (wmISH) is a powerful tool to visualize the gene transcript in embryonic tissue. This study aimed at setting up wmISH using digoxygenine-labled RALDH2 antisense riboprobe.Methods & Materials: The early developing chick embryos were fixed and dehydrated upon dissecting. Following bleaching and protein kinase K treatment, the embryos were hybridized with hybridization buffer and then with the buffer containing RALDH2 antisense riboprobe for 36h. Upon washing with SSC solution and incubation with anti-digoxygenin Ab, the embryos were treated with NBT/BCIP and transferred to NTMT buffer. Some embryos received sense ribobrobe and negative control was considered as a group not treated with any riboprobes. Following binding the antisense riboprobe to RALDH2 transcript, blue color appeared in somites and neural tube as well as the cardiac tube. No color was detected in the embryonic tissue treated with the sense riboprobe or in untreated group.

In vitro investigation of hydroethanolic extraction of Cirsium vulgare on growth rates of neonate rat neural stem cells. Neural stem cells were isolated from hippocampus of neonatal rat brain. To determine optimal concentration of Cirsium vulgare extraction، isolated neural stem cells were treated with 200، 400، 600، 800 and 1000 µg/ml for 48 h and then cells proliferation rate were evaluated by MTT assay. In addition Sox2 mRNA expression in neural stem cells was evaluated using quantitative real-time PCR. The results of this study showed that the Cirsium vulgare extract caused significant increase in cell proliferative activity and Sox2 mRNA expression when compared with the control group. with respect to the effect of Cirsium vulgare extract on the proliferation rate of neural stem cells، the use of hydroethanolic extraction of Cirsium vulgare can be used for clinical purposes to treat some of the neurodegenerative disorders such as ischemic stroke and spinal cord injury.

Nerve tissue engineering (NTE) is one of the most promising methods for the treatment of the central nervous system (CNS) neurodegenerative diseases. The three-dimensional distribution and growth of the cells within the porous of the scaffold have a significance clinical role in the NTE field. Scaffolds used in tissue engineering, not only must have a good performance, but they should also be porous, biocompatible and biodegradable. The present work aimed to fabricate and study the morphology, biodegradability and chemical characteristics of Poly-L-Lactic-Acid (PLLA) in order to use in the neural tissue engineering. In this experimental study, PLLA nano scaffold was fabricated with an appropriate structure and morphology using Electrospinning Technique. Fourier Transform Infrared (FTIR) spectroscopy and Scanning Electron Microscopy (SEM) were used to determine the physico-chemical properties of the scaffold. Scaffold biodegradation was studied in Phosphate-buffered saline (PBS) for 40 days. Isolated stem and progenitor cells from subventricular zone of the adult mouse brain were cultured on the scaffold and their morphology and connection properties were characterized using SEM. SEM studies indicated that PLLA is a nano-fibrous scaffold which shows the appropriate surface characteristics. Furthermore, this nanoscaffold showed a high degradation and water uptake rate in the degradation test. Finally, SEM studies confirmed the attachment and growth of the mouse neural stem and progenitor cells on the scaffold. These results suggested that the PLLA nano scaffold is an appropriate structure for the growth and differentiation of the neural stem and progenitor cells and the electrospining technique is an efficient method for the scaffold producing used in the nerve tissue engineering.

Background and Natural bone is a combination of polymer and biological apatite, therefore, the composite scaffolds made of polymers and bioactive ceramics have found wide applications in bone tissue engineering studies. Among various polymers, the poly-L-lactic acid (PLLA) and hydroxyapatite (HA) have attracted much attention due to their optimal properties. In this study, using PLLA polymer and hydroxyapatite (which is similar to human bone mineral component) three-dimensional composite scaffolds were developed by Electrospinning Method. Then the behavior of human umbilical cord mesenchymal stem cells (MSCs) was investigated on the scaffolds. The aim of this research was to develop an appropriate bioactive and functional scaffold for bone tissue engineering. In this study mesenchymal stem cells were isolated from the human umbilical cord. The cells were cultured on both PLLA and PLLA/HA (10%) composite polymeric nano scaffolds. Biocompatibility of scaffolds was confirmed by MTT assay. The morphological and cell adhesion characteristics of MSCs on the scaffolds were compared using Scanning Electron Microscopic (SEM) imaging. Finally, the cells were treated with osteogenic differentiation medium for 21 days in order to investigate their differentiation potential on the scaffolds. The differentiation of the stained cells with Alizarin Red and Von Kossa were studied at 7,14 and 21 days after cultivation. SEM studies showed that the surface properties of both scaffolds were desirable and the cells not only had the ability to attach and proliferate better on the nanocomposite scaffolds, but were also in a natural condition morphologically. The comparison of staining results indicated a higher differentiation rate in composite polymeric nano scaffold. The results showed that the PLLA/HA nano scaffold could be a very good candidate for bone tissue engineering.

Different processing methods are being used to improve the quality of hematopoietic stem cell transplantation. Using hydroxyethyl starch, simple centrifugation and Sepax automation, this study was aimed to compare these three conventional methods. 90 cord blood samples were taken and processed by hydroxyethyl starch, simple centrifugation and Sepax automation methods. Then they were subjected to total nucleated cell (TNC) counting and CD34 positive counting as well as colony assay. Finally, all data were analyzed using one-way analysis of variance (ANOVA) and ps less than 0.05 were considered statistically significant. The TNC recoveries in hydroxyethyl starch, simple centrifugation and Sepax automation methods were 76%, 71% and 80%, respectively (p> 0.05). The CD34+ cell recoveries in the Sepax automation and in the other two methods were 91% and 85%, respectively (p> 0.05). Also, the colony assay recoveries were not significantly different among the three methods (p> 0.05). No significant difference was seen in TNC number, CD34 positive counting and colony formation among the three different methods.

Background and Umbilical cord derived Wharton's jelly is an enriched and accessible ‎source of stem cells with highly proliferative and differentiation potential. This study aimed to evaluate the ‎surface markers and related genes of the stem cells isolated from the human Wharton's jelly.‎ Explants of the human umbilical cord derived Wharton's jelly was dissected ‎and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% Fetal bovine serum ‎‎(FBS). Then, those cells migrated from explant's boundary were replated and passaged in DMEM containing ‎‎10% FBS. Finally, by using flowcytometry and (Reverse–transcriptase polymerase chain reaction) RT-PCR ‎techniques different surface markers and related genes were analyzed.‎ 5-7 days post-plating, the stem cells initiated the migration from cultured explants and ‎showed up to 80% densities on days 16-18. Light microscopy demonstrated two distinct cell populations ‎including fibroblast-like and flat endothelial-like cells. Dual staining with flowcytometry also revealed that ‎the cultured cells were found to be positive for CD44, CD73, CD90, CD105 and negative for CD34 and CD45. ‎RT-PCR showed no changes in CD marker expression pattern during different passages.‎ Human Wharton's jelly derived stem cells appear mesenchymal cell morphology and ‎express related surface markers but no hematopoietic stem cell markers.‎

The somites are epithelial blocks of mesodermal cells differentiating to sclerotome and dermomyotome and notochord or axial mesoderm plays a major role in somitic cell differentiation to sclerotome. This study was aimed to evaluate the role of the notochord in sclerotomal differentiation of the somites in vitro. In this experimental study, isolated notochords from chick embryo were encapsulated in alginate beads and co-cultured with somites for six days. In control group, the somites were cultured alone. Newly isolated and non-cultured somites were considered as somites on day 0. Eventually, morphology of cultured somitic cells was evaluated by inverted microscope and then RT-PCR method was used to analyze the sclerotomal and dermomyotomal gene expression profile. In comparison with control group, the somitic cells co-cultured with notochord had highly differentiation potential and showed mesenchymal morphology with numerous slender processes. Gene expression profile of co-cultured somitic cells indicated upregulation of Pax1 and BMP4 and downregulation of MyoD, presenting sclerotomal cell characteristics. Embryonic notochord can induce in vitro sclerotomal differentiation in somites through upregulation of genes involving in this differentiation.

Retinoic acid (RA) is a vitamin A derivative and one of the most important inducing signals in vertebrates that is involved in differentiation، morphogenesis، apoptosis، and reproduction. This study was done to evaluate the role of RA in in vitro neural patterning of mouse embryonic stem cells (mESCs). In this experimental study، upon formation، embryoid bodies (EBs) from mESCs، Royan B1، were induced by 1 µM RA for four days and then plated for eight days. Untreated EBs were considered as the control group. Finally، in both groups، neural induction and patterning of EB-derived neural cells were evaluated by using immunostaining، flowcytometry، and RT-PCR methods. RA induced neurogenesis in ES cells، from which 35% showed to express MAP2. RT-PCR analysis also indicated that RA-treated neural cells derived from ES cells could at the same time express Mash1، Pax6/7، and Dbx1/2 as dorso-ventral (DV) pattering markers and Hoxb4، Hoxc5، and Hoxc8 as the rostro-caudal (RC) axis markers. RA induces in vitro neural induction along with neural patterning of ES-derived neural cells in DV and RC axes. Keywords: Mouse embryonic stem cells، neural patterning، retinoic acid

Stem cells are a class of undifferentiated cells that are able to differentiate into specialized cell types. The discovery of such cells in the adult mammalian central nervous system (CNS), an organ traditionally thought to have little or no regenerative capacity, opened the door to treatment of degenerative diseases of CNS like Stroke, Parkinson, Alzheimer and Spinal Cord Injury. Thus, here we described the isolation of neural stem cells from the adult mouse brain using the neurosphere assay (NSA) and differentiation of these cells to neural adult cells in details. The rostral part of the subventricular zone (SVZ) of the lateral ventricles in the adult mice was dissociated into single cell suspension and cultured using NSA. Primary neurospheres were counted seven days after plating and then the mean number of neurospheres was recorded. The differentiation of neural stem cells into adult neural cells was accomplished by plating the neurosphere-derived cells in differentiating media. Immunocytochemistry and specific markers were used for the identification of the adult neural stem cells. The cell suspension obtained from the rostral part of the SVZ of the lateral ventricles generated multipotential colonies, called neurospheres, 7 to 10 days post- incubation. The mean number of neurospheres generated from SVZ was 505±62. The multipotentiality of the neurospheres was shown by palting them in differentiating media and generating adult neural cells including neuron, astrocyte and oligodendrocyte. Owing to their rarity and paucity of neural stem cell specific markers, the NSA is a common and selective method for isolating and understanding the biology of embryonic and adult neural stem cells.

The aim of the present study was to understand if EBs can promote generation of neurons following co-culture with chick embryo somites.

The mouse ES cells line Royan B1 were cultured in hanging drops to induce embryoid bodies (EBs) formation. Then RA was added to some EBs according to 2-/2+/2+ protocol to evaluate the potential of their neuron generation. Somites were isolated from the chick embryos and then embedded in alginate solution. Finally alginate beads containing somites were co-cultured with EBs as 1:1 and 4:1 ratio.

Mean percentage of EBs containing neurons in RA somite 4:1 somite 1:1 and control were 82.8% 35.3% 21.1% and 4.8% respectively. Our results showed that EBs can promote generation of neurons following co-culture with somites and somite 4:1 had more profound effect on neural induction compared to somite1:1. The neurons were formed rapidly in RA and somites groups than control group. The somite groups induced higher rosette formation which had the capacity to generate neurons upon isolation and replating. Finally the neural properties were confirmed by immunocytochemistry procedures.

Chick embryonic somites can induce ES cells –derived EBs to generate neurons in vitro and may lead to formation of rosette structures with neuron formation capacity.

Considering the high prevalance of congenital obstruction of nasolacrimal ducts in infants and the importance of timely intervention and due to the lack of previous similar researches in Ardebil province, and regarding the discrepancies in the reports about the degree of successful accomplishment of the first probing, the researchers set out to design and perform the present research among the patients referring to ophthalmology ward. This descriptive and cross-sectional study was conducted on 84 children suffering from congenital nasolacrimal duct obstruction who had undergone pobing surgery in Alavi hospitaly between 2000 and 2002. 70 eyes of 50 patients were examined. 27 (54%) were male and 23 (46%) were female. 20 of them (40%) had bilateral involvement and 30 (60%) had unilateral one. Involvement of right eye (60%) was more than left eye (40%) and was expected in first child (56%) more than subsequent offsprings. Clinical manifestation was epiphora in 44%, prulent discharge in 12% and epiphora with prulent discharge in 44%. 90% of the subjects were operated for the first time while 10% had had 2-3 operations before complete recovery was observed in 48% of the patients 46% of them had relative improvement and no recovery was witnessed in 6% of them. This study showed that the male infants were affected more than females, and the rate of involvement in right eye as well as in first children was relarively high. Also the rate of recovery among unilateral groups and infants under 1 year of age was considerably high, so it is recommended that the patients of this age group undertake a therapy with topical antibiotic and lacrimal sac massage and because of the low success of the surgery above 2 years of age, probing is recommended from 6 month to 18 months of age if medical treatment was unsuccessful.

Infection with Toxoplasma gondii can cause severe illness when transmitting to fetus or when it is reactivated in immune-suppressed persons. The aim of this study was to determine the prevalence of antibodies against toxoplasma gondii in women referring to laboratory of health center for medical examinations before marriage. This descriptive cross-sectional study was performed on 504 samples collected from women in Arabil, Iran, in 2002. The samples were studied by indirect immunofluorescent assay (IFA) for determination of IgG and IgM antibodies to toxoplasma. The seroprevalence of IgG antibody at a titer of ³ 1:20 was 34.7%. The highest antibody titer frequency was observed in 1:20 titer (11.7%) and the lowest belonged to 1:3200 (0.4%) and 1:6400 (0.4%) titers. 20 persons (4%) showed IgM antibody against Toxoplama gondii. No statistically significant differences were observed between the prevalence of antibodies on the one hand and age and history of contact with cat or domestic animals on the other. since 65.3% of these women in Ardabil were seronegative, health education is required to omit the risk factors, especially during the pregnancy.

Nutritional status of individuals and community are influenced by different factors. Investigating nutrition and food problems as well as determining dietary pattern play a crucial role in determining nutrition programs and policies, preventing food deficiency, improving nutrition level and preventing malnutrition diseases. The aim of this study was to determine calorie and nutrients intake, food habit and dietary pattern in rural areas of Ardabil. In this cross-sectional study 250 families from 15 rural areas of Ardabil were selected using simple random method. Nutrition status of all the individuals was studied using 24 hour recall and food frequency. Data were analyzed using SPSS and Food processor. This study showed that intake of some nutrients (Zinc, Selenium, Folic acid and Vitamin B2) in the subjects were significantly less than Recommended Dietary Allowance (RDA) of WHO (P<0.001), but intake of protein, iron and calcium was more than the RDA of WHO (P<0.001). Calorie intake of 20% of rural people was less than 75% of RDA(mostly seen among men). The findings of food frequency showed that main foods of rural individuals in Ardabil were local bread (Lavash), potato, egg, milk, biscuit, yogurt, garlic, onion, vegetable fat, butter and tomato. This study showed unsuitable intake of some nutrients. In order to improve the nutritional status of these people, regular intake of different groups of foods should be instructed.

Nutritionists have traditionally recognized breakfast as the most important meal of the day. The importance of eating breakfast is for growing and nutritional well being of children. While health habits such as eating breakfast, maintaining weight, and sleeping regularly are related to the longevity of adults, very little is known about the health habits of disadvantaged school-age children. This study set out to evaluate eating breakfast among adolescent girls and its relationship with body mass index, weight and nutrient intake. This descriptive cross-sectional study was done on 611 adolescent primary and secondary school girls (10-14 years old) in Ardabil. Anthropometric studies (height, weight, and MAC) and nutritional status (24 hour recall and food frequency) were done, and the students were asked about eating breakfast and other snacks. The collected data were analyzed using SPSS ver. 9 and Food Processor. About 16.85% of girls had come to school without eating breakfast. BMI and body weight of these students were significantly more than those of girls who ate breakfast (P<0.05). On the other hand snack intake among the girls who did not use to eat breakfast was more than that among breakfast eaters (P<0.05). The amount of calorie, protein, thiamin, niacin, calcium and iron intake in breakfast-eating girls was more than those among non-eaters (P<0.05). The amount of folacin, riboflavin, calcium and zinc intake in non-eaters was less than recommended dietary allowance of WHO. The result of food frequency showed that the consumption of food such as protein different types of bread, potato, and legume among breakfast eaters was more than their consumption among non-eaters. This study indicated that eating breakfast can provide adequate calorie and nutrients but its omission can not only lead to lower calorie and nutrients intake but also have an unfavorable effect on food habit and weight of the students.

Menarche is an important event during developing stages in females. Several factors can affect the determination of the menarche age including socioeconomic situation, family customs and nutritional status. Different researches show that obese girls become menarche earlier than thin ones. Regarding the importance of nutrition in menarche age, the present study was conducted to evaluate nutritional status among girls in Ardabil at menarche age (10-14 years old). The aim of this study was to determine the relationship among anthropometrics (e.g. weight, height, BMI, MAC), nutritional status, economic factors and menarche age. This cross - sectional study was done on 612 primary and secondary school girls at the age of 10-14 during 1999-2000. Height, weight and MAC of these subjects as well as their BMI were calculated. Their nutritional status (24-hour recall and food frequency) and menarche age were studied. Finally, the data were analyzed by SPSS ver.9 and Food Processor. There was a significant relation between height and menarche age (P<0.05). Menarche age reduced with increasing of weight and BMI, and the differences were significant (p<0.05). Menarche age of girls whose MAC was less than 22cm was lower than individuals with MAC≥22 cm and this difference was significant (P<0.01). Girls with≥40% caloric intake from fat had lower menarche age than the other groups but this difference was not significant. Food frequencies showed that the most frequently used foods in each week were two kinds of local bread (Lavash and Barbary), sugar, vegetable oil, chocolate, cheese, apple and rice. Menarche is affected by anthropometrical factors and nutritional status (particularly fat intake).