مجله سلول و بافت
سال سیزدهم شماره 4 (زمستان 1401)

Optimizing cell culture conditions to improve oocyte growth and maturation in vitro culture conditions has been the focus of many researchers today, but the quality improvement mechanism is not fully understood. Apoptosis or programmed cell death is a process to remove old and damaged cells from tissues and plays a major role in the life of follicles and immature oocytes. Most of the defective reproductive cells as well as extra cells are removed from the ovaries through apoptosis. One of the ways to reduce oxidative stress in the laboratory culture of oocyte maturation is the use of antioxidants. Statins are drugs for the treatment of high cholesterol for which antioxidant and anti-apoptotic properties have been reported. Atorvastatin in high doses has side effects for the heart and kidneys, but in low concentrations, it has antioxidant and anti-inflammatory effects for cells, and no side effects have been reported for it.In this study, we investigated the effect of low-dose atorvastatin on the rate of apoptosis in mouse oocytes. 

24 h after PMSG (Pregnant Mare Serum Gonadotropin) inhection, 200 oocytes were obtained from adult female Wistar rats at the age of 4-5 weeks, were from were divided into 2 groups of 100 including the control group (MEM:Minimum Essential Medium culture+ Growth factor) and the atorvastatin group (MEM culture medium + 2 mg/kg atorvastatin+Growth factor) and cultured in the incubator(35 oC,CO2 %). After 24 hours of oocyte culture, Matured oocytes that met the standards of a healthy mature oocyte(oocytes that were in the first meiosis and had the first and mature polar body) were isolated the amount of apoptosis in each group was checked using tunnel staining and fluorescent microscope, the data were analyzed by variance analysis.

After 24 hours of oocyte culture, the amount of apoptosis in the group receiving atorvastatin in the culture medium and the control group was investigated. The rate of apoptosis in the atorvastatin group and the control group was 24% and 22%, respectively, In the atorvastatin group, apoptosis increased by 2% compared to the control group butt the difference between the two groups was not statistically significant (p =0.11).

According to the findings of this study, atorvastatin in low doses can have a pro-apoptotic effect and cause partial induction of apoptosis in mouse oocytes in a laboratory environment. Although this study was not conducted on other doses of atorvastatin, it is suggested that the effect of other doses of this drug on the rate of apoptosis induction should be investigated in order to make an evidence-based decision regarding its administration and use.

Long non-coding RNAs (lncRNAs) are a group of RNAs known as key regulators in cancer. The role of these molecules in differenet cellular procecess including cellular growth and differentiation has been documented. Several non-coding RNAs have been identified in the cell. Among the LncRNAs, LnvRNA MIAT is a non-coding RNA whose oncogenic expression is known in different types of cancer. However, uts role in brain tumors in not fully understood. Due to its oncogenic role in other cancers, it is estimated that this molecule to play an important role in brain tumors as well. The aim of this study was to investigate the role of lncRNA MIAT in glioma and identify its regulatory mechanism.

In this study, the expression of lncRNA MIAT in glioma cancer cells, U87-MG and A172, was evaluated by Quantitative Real-Time PCR (qRT-PCR) method. Then, its expression was inhibited by the RNA interference method, and the expression of CD44 and miRNA-302 target genes was investigated.

The results showed that lncRNA MIAT was highly expressed in glioma cancer cells. Also, after inhibition of the expression of lncRNA MIAT, a decrease in the expression of CD44 and miRNA-302 was observed.

The findings suggested the oncogenic role of lncRNA MIAT in glioma cancer cells through the regulation of CD44/miRNA-302 expression. As a result, lncRNA MIAT could be evaluated as a new biomarker in the prognosis and diagnosis of glioma.

Cartilage tissue has limited capacity for spontaneous repair and self-renew due to the absence of vascularization and progenitor cells, thus requiring a therapeutic approach to repair tissue damage. Recently, extracellular vesicles (EVs) have attracted attentions because of their major roles in cell communication and tissue repair by regulating cellular processes such as cell growth, differentiation, and proliferation. Therefore, it is necessary to isolate extracellular vesicles with chondrogenic potential from an appropriate cellular source for cartilage regeneration. This study aims to compare the chondrogenic ability of extracellular vesicles derived from cocultured -chondrocytes / mesenchymal stem cells (CHO / MSC) at ratios of 1/2 and 1/4.

Towards this goal, chondrocytes and mesenchymal stem cells were isolated from rabbit articular cartilage and bone marrow, respectively. Mesenchymal phenotype of isolated MSCs were characterized based on their surface markers and by differentiation into mesenchymal lineages. Chondrocytes and mesenchymal stem cells were co-cultured with defined ratios, their conditioned media were collected and extracellular vesicles were extracted with an ultracentrifuge. Concentrations of extracellular vesicles were determined using BCA Protein Assay Kit, then EVs were characterized in terms of size, morphology, and expression of surface markers by dynamic light scattering (DLS), scanning electron microscope (SEM) and western blotting, respectively. Mesenchymal stem cells were treated with different concentrations of extracellular vesicles (50, 100, and 150 ug/ml) for 21 days. Quantitative real time-PCR (qRT-PCR) and histological analysis were subsequently performed to assess the quality of chondrogenic differentiation among experimental groups. The differentiation of MSCs to osteogenic and adipogenic lineages  was demonstrated by Oil Red and Alizarin Red staining after 21 days.

The results of flow cytometry showed that CD90 was expressed by 88.6% of the cell population as a positive marker and CD34 as a negative marker was only expressed in 5.48% of the cell population. SEM micrographs confirmed the spherical shape of CHO/ MSC-EV 1/2 and CHO/MSC-EV 1/4 and the mean particle size of isolated EVs were 51.66 ± 8.15 and 19.11 ± 6.03 nm, respectively. Special surface markers for extracellular vesicles containing CD9 and CD81 were expressed in both groups. At concentrations of 100 and 150 ug/ml of CHO/MSC-EV 1/2, higher cartilage - specific markers, including Col II were observed compared to CHO/MSC-EV 1/4. Similarly, safranin O and toluidine blue staining revealed the more deposition of aminoglycan and proteoglycan at concentrations of 100 and 150 ug/ml of CHO/ MSC-EV 1/2, compared to CHO/ MSC-EV 1/4. This study demonstrated the chondrogenesis potential of extracellular vesicles, especially in CHO /MSC-EV 1/2 and extracellular vesicles derived from co-culturing chondrocytes/MSCs improve matrix production and chondrogenesis.

Our research shows that chondrocyte-MSCs proximity is essential for the response as improved viability, chondrogenesis, and matrix formation in recipient MSCs. It is also proposed that co-cultured derived EVs with the ability to promote chondrogenesis have the potential to be utilized in cartilage regeneration.  Due to their immunogenicity and their low tumorigenic potential, extracellular vesicles can be used to help repair cartilage tissue.

In thyme, thymol and carvacrol have attracted the attention of pharmacologists due to their various therapeutic properties such as anti-tumor activity. To date, a variety of treatments such as elicitors have been proposed to increase the content of thymol and carvacrol. This study was also conducted with the aim of evaluating the synergistic effect of UV-A ray and methyl jasmonate on the biosynthesis of thymol and carvacrol.

Thyme seeds were planted in plastic pots at a factorial experiment in time with a completely randomized design with three replications under greenhouse conditions. The five-leaf seedlings were separately and simultaneously irradiated with UV-A and sprayed with 0.1 mM methyl jasmonate. After 24 and 48 h of treatment, the expression levels of GTS, DXR, CYP180, and CYP178 genes were measured by Real-Time PCR and the metabolite levels were measured by HPLC.

The expression of GTS, DXR, CYP180, and CYP178 genes was affected by the synergistic impact of methyl jasmonate and UV-A. The transcript level of these genes increased significantly after 24 h of individual treatments and this increase was more in the combined treatment of hormone and radiation. However, the expression of these genes was linked with a significant decrease after 48 h of treatment. The increasing and decreasing trend of thymol and carvacrol content in the elicitor treatments was also in accordance with the expression of studied genes.

Our observations suggest that thyme uses synergistic signaling to increase its metabolite levels during the first 24 h of hormone and radiation treatments. And after that, other defense mechanisms are activated and then the level of secondary metabolites decreases.

Cancer is one of the most important diseases of this century, which affects many people all over the world, and while the prevalence of this disease is increasing, no suitable treatment has been found yet. In the meantime, breast cancer is the most common cancer and the leading cause of cancer-related death in women worldwide. In the search for cancer treatment solutions, researchers synthesize numerous medicinal compounds for various cancers and study their effects. Sorafenib is a urea multikinase inhibitor which causes apoptosis and inhibits angiogenesis and cancer cell proliferation. This compound is currently used as a treatment for hepatocellular cancer, and tests have also been carried out to verify its performance in treating other cancers. Sorafenib derivatives were also synthesized and analyzed. In this research, the anticancer effect of one of the derivatives of sorafenib named (2E,2´E)-2,2´-(1,4-phenylene bis(methanylidene)bis(N-(4-chloro-3-(tri) Fluoroethyl (phenyl) hydrazine carboxamide, abbreviated as SO-D-3, was evaluated against three breast cancer cell lines.

Three breast cancer cell lines including MCF7, 4T1 and MDA were prepared and cultured in RMPI culture medium. Cells were treated with concentrations of 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM of SO-D-3, and their survival rates were measured using the MTT test during 24, 48 and 72 hours. The induction of apoptosis by SO-D-3 in the studied cancer cells was investigated by flow cytometry. In addition, the level of Hsp70 and Casp8 proteins as implicated in apoptosis were investigated using the western blot method. The results were statistically analyzed using SPSS software.

The results of the MTT test showed that the treatment of cancer cells significantly decreases the survival of these cells compared to the control. The IC50 for MDA-MB-231 cells was significantly lower than MCF7 and 4T1 cells at 24 hours (157.4 µM for MDA-MB-231 compared to 843.8 µM for MCF7 and 1212 µM for 4T1). However, the difference decreased with increasing treatment time (77.47 μM and 23.38 μM for MDA-MB-231, 107.3 μM and 36.69 μM for MCF7, and 83.63 μM and 16.58 μM for 4T1 at 48 hours and 72 hours respectively). Flow cytometry analysis revealed that in cells treated with SO-D-3, apoptotic cells increased significantly. Examination of the protein level using the western blot technique also showed that the HSP70 protein was significantly decreased in MCF7 and 4T1 cells. On the other hand, the level of Casp8 protein was significantly increased in 4T1 and MDA cells.

The results of the present study showed that SO-D-3 can cause the death of breast cancer cells in a time-dependent manner. Subsequent experiments showed that SO-D-3 exerts this action through the induction of apoptosis. Therefore, according to the results of the present research, SO-D-3 can be introduced as a compound with potential anti-cancer properties that can cause cell death by reducing Hsp70 and changing the expression of Casp8 involved in the external pathway of apoptosis.

Infertility is a life crisis that affects patients worldwide. Infertility is defined as failure to conceive after 12 months of sexual activity, affecting 15-17% of couples worldwide. and about 50% of them are related to the factors of female infertility. Activating the process of meiosis in the oocyte and its maturation has been one of the important therapeutic goals of infertility researchers. Today, inducing oocyte growth and development outside the body is one of the methods used in assisted reproduction technology. Bone marrow is a complex organ in which different lineages of hematopoietic and stromal cells support hematopoiesis. Extracellular vesicles (EVs) with a size of 20-100 nm are released by different types of cells in culture media. In addition to proteins, exosomes are enriched with an array of cytokines, specific lipid rafts such as phosphoglyceride, cholesterol, ceramide, fatty-acyl chains, as well as mRNAs, miRNAs, non-coding RNAs, tRNAs, rRNAs, and rarely DNA.  The purpose of this experimental research is to investigate the effect of exosomes derived from the bone marrow stem cells of small laboratory mice on the maturation of preantral follicles.

Exosomes were isolated and cultured from the mesenchymal stem cells of the bone marrow of small laboratory mice by the flushing method. Identification of stem cells was done by flow cytometry method and separation and purification of exosomes was done by ultracentrifuge, identification of exosome was also checked by atomic force microscope (AFM). The effects of exosomes on the viability of follicles were measured by the MTT method, and developmental parameters such as the diameter and the formation of the antrum cavity in the follicles were examined and the follicles were examined on days 0, 2, 3 and 4 of culture with 20 magnification and inverted microscope. Photographs were taken and the diameter of the follicles was measured in micrometers by Image J software. The expression of GDF-9, BMP-15 and BMP-7 genes as genes involved in the growth and maturation of follicles was investigated using Real Time-PCR method. GraphPad Prism 8 software and one way Anova statistical test were used to analyze the data of this research.

The evaluation of the viability of the follicles showed that compared to the control group, the follicles treated with exosome 25, 50 and 100 micrograms/ml showed an increase in viability, as well as the rate of antrum formation increased significantly in the group with the concentration 100 μg/ml showed a significant level (p<0.01**) compared to the control group. The diameter of the follicles increased with increasing the concentration of exosomes compared to the control group. GDF-9, BPM-15 and BMP-7 genes also increased in the treatment groups.

According to the findings of this experimental research, it can be stated that exosomes derived from bone marrow stem cells Small laboratory mice have a positive effect on survival and maturity, as well as the growth of ovarian follicles.