@2024 Afarand., IRAN

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ISSN: 2252-0805    The Horizon of Medical Sciences 2018;24(4):270-276

Background
Sativo cannabis is from flowering plants and the Cannabaceae family. Cannabaceae consists of two genera and cannabis is considered as a single species of Cannabaceae family [1].
Previous Studies
... [2]. The cannabis, as one of the contents of the cannabis plant, is used as the most commonly used psychoactive substance by million people throughout worldwide [3]. ... [4]. The cannabis extract has apoptotic effects that can be useful in diseases, such as cancer [6,5]. Also, the mutagenic and teratogenic effects of cannabis compounds have been considered by researchers and scientists have carried out research on the chromosomal damage of cannabis users [7]. ... [8-10]. Stem cells are non-specialized cells generating tissues in the human body. Therefore, they are appropriate candidates for therapeutic use in tissue regeneration and repair. Stem cells are defined as ancestral or progressive cells as well as clonogenic cells capable of regeneration and differentiation into several tissues [11]. ... [12-14].
Aim(s)
The aim of this study was to analyze the effects of marijuana on differentiation potential of bone marrow stem cells in adult male rats to adipose tissues.
Research type
This research is an experimental study.
Research society, place and time
This experimental study was conducted on the rats in the Islamic Azad University, Shiraz branch in 2017.
Sampling method and number
This experimental study was performed on 5 rats.
Used devices&materials
The protocol of this study was approved based on international standards for laboratory animals and by the Ethics Committee of the University (Ethics code. IR.miau.REC.1396.807). In this study, the hydroalcoholic extract of cannabis was prepared by percolation method to study the effect of cannabis on differentiation of bone marrow mesenchymal stem cells in rats into adipose tissue cells. In this study, 5 male adult male rats aged two months weighing approximately 180-200 g were purchased from the animal house of the Comparative and Experimental Medical Center of Shiraz University of Medical Sciences to obtain bone marrow mesenchymal cells. They were transferred to the Animals Laboratory at the Muhammad Rasoololah Research Tower of the Shiraz University of Medical Sciences. After two hours, using inhalation method, animals were transferred to a tray under the laminar hood (JAL TAJHIZ-JTLVC2X-IRAN) and the hip and tibia bones were separated and covered by sterile Falcon (BD-USA) containing saline buffered phosphate and penicillin-streptomycin antibiotic (1%) and antifungal amphotericin. In order to prevent Falcon tissue damage, the contents of the sample were placed on ice and two ends of the bones were cut under complete sterile conditions. Then, the bone marrow was washed with a syringe using F12 DMEM (Bio idea, Iran) without FBS from the two ends of the bone. Cell suspension of the Falcon was Centrifuged (Eppendorf Centrifuge 5702R-Germany) at 1000 rpm at 20°C for 5 min. The cell mass residue at the bottom of the tubes plus suspension culture F12 DMEM (1 ml) was transferred to a Flask 75(Orange, USA) and cultured with 15 ml F12 DMEM containing 15% FBS (GIBCO, USA). The cells were then incubated at 37°C with 5% Co2. When the cell density reached 80%, a cell passage was performed and the cells were used for culture after the third passage. In this study, spindle-shaped and fibroblast-like morphology of the cells were examined using the inverted microscopic (Nikon ECL IPSE TS100-JAPAN) and their mesenchymal function was confirmed. In this study, MTT test was used to measure the toxicity of hydroalcoholic extract of cannabis and determine its acceptable amount to use. Two other plates were changed within 72 h, one plate changed to the medium without extract every 24 h in all wells and the other plate experienced the increasing dosage of the extract to the medium as the first day. Both plates were tested by MTT after 72 h. In this study, based on the results of MTT test, 30-150 ng/ml dosage was selected as the favorable concentration, among which the optimum dose of 100 ng/ml was used to treat the cells. In this study, expression of some specific genes in stem cells was examined by RT-PCR. mRNA extraction, cDNA production by PCR and the proliferation of gene associated with CD34 and CD45 as the markers of hematopoietic cells, and also proliferation of gene associated with CD73 and CD90 as the specific markers of stem cells were performed to investigate cells surface markers. Moreover, 6-well cultures were used to determine the differentiation of bone marrow mesenchymal cells into adipose cells and each well was fulfilled about 70-80%. Then two wells were regarded as negative controls (DMEM + FBS10% culture medium), two wells as positive control (adipose differentiation medium) and two wells as experimental medium with ascorbic 3-phosphate (50 μg/ml) (Merck; Germany), dexamethasone (100 nmol) (Sigma Aldrich; Germany), indomethacin (50 μg/ml) (Sigma Aldrich; germany) and cannabis extract. The plates were kept in incubator (CO2 Incubator-MEMMERT GERMANY) for 21 days and the wells were changed every two days. To evaluate cannabis sativa extract-induced differentiation to adipose cells, bone marrow mesenchymal stem cells 21 days after treatment with the extract, all wells were fixed for 45 min with formalin 4% (Merck; Germany). They were washed with alcohol 70% (w/v) (Merck-germany) were stained with Oil Red (Bio Idea; Iran). The cells then were washed three to four times with alcohol 70% (w/v) and examined by Inverted microscope (Nikon ECL IPSE TS100-JAPAN). One-way ANOVA and Scheffe's post-hoc tests using SSPS 23 were employed.
Findings by Text
Most of the extracted cells in the flasks were non-adherent and separated during the first washing of the flask and culture medium change. Some of the remaining cells were colonized and after about 3-4 days after culture, a single-cell layer with a density of 80 to 90% was formed in the flask. After treating the cells with trypsin, the cells were again rounded and returned to the flask. Three days later, a major part of the culture medium was filled with spindle-shaped mesenchymal stem cells. The growth and proliferation of the cells became more rapid following the passage 1 and the subsequent passages, so that within 3-4 days, 80-90% of the culture was covered by the cells. In the third passage, most of the cells had spindle-shaped and fibroblast-like morphology, which is a common form of mesenchymal stem cells. In addition, the specific bond of mesenchymal stem cells was observed after RNA extraction, cDNA production and the polymerase chain reaction. In addition, no specific bond related to hematopoietic stem cells (Figure 1). Treatment with cannabis extract not only do not change the appearance of mesenchymal stem cells, but also the results of this study indicated an increase in the survival of treated cells compared with control group cells. No significant difference was observed between the survival of the cells in the groups receiving different doses of cannabis extract (Charts 1 and 2). Daily treatment with 100 ng/ml of the cannabis extract to the differentiation culture of the experimental group cells resulted in forming first adipose lineages as the control group cells, so, after 10 days, the cytoplasm of the cells was filled with adipose cells. After 21 days, using Oil Red staining method, the adipose nature of the lineages observed in the cytoplasm of the control and experimental groups was confirmed (Figure 2)
Main comparison to the similar studies
The cannabis extract at the used concentration had no effect on the attachment to the culture flask or cells appearances, which is consistent with the previous studies to prove mesenchymal function of stem cells [15, 16]. .... [17-19]. In this study, cannabis was not an inhibitor of differentiation to adipose cells, and the results are consistent with the studies to make sure about mesenchymal stem cells by their differentiation into adipose cells [20]. ... [21-29].
Suggestions
None declared.
Limitations
None declared.
Conclusions
The results of this study revealed that marijuana has positive effects on mesenchymal bone marrow stem cells differentiation to the adipose cells.
Acknowledgments
Authors are thankful to the efforts and assistance of the Stem Cell Research Center of the Shiraz University of Medical Sciences, who cooperated in this research.
Conflict of interest
None declared.
Ethical Permissions
None declared.
Funding Sources
None declared.

TABLES and CHARTS

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