Study On The Differentiation Of Bone Marrow Mesenchymal Stem Cells Of Rat Induced By Astragalus Mongholicus And Its Mechanisms

Degenerative diseases of the central nervous system (ND) are a group of chronic and progressive nervous systemic diseases based on the primary neuronal degeneration. They mainly include Alzheimer disease, Parkinson disease, Huntington disease etc. and they have the common features: neuronal degeneration and apoptosis. Despite a lot of progression of researches on ND, there are no effective therapia and drugs to prevent and cure the diseases.At present, the researches on stem cells bring the hopes for the cure of ND. Stem cells are defined as cells with two main properties: self-renewal capacity, and commitment to multi-lineage differentiation. According to the source and differentiation potency, stem cells can be divided into totipotent stem cells, pluripotent stem cells and multipotent stem cells (adult stem cells) and so on.The researches on embryonic stem cells (ESC) have made more progresses. People expect that it will play an important role for curing some diseases. Because of its lack of resources and the problem of social ethics, the clinical application of ESC will face more difficulties. Now, some researchers have aimed at bone marrow mesenchymal stem cells (BMSCs). In recent years, several studies have showed that bone marrow mesenchymal stem cells have derived from adult bone marrow, which do not belong to hematopoietic tissues, but provide a physical support for maturing hematopoietic stem cell, secrete many kinds of growth factors and cytokines controlling differentiation and maturation of them. Under proper environment, BMSCs can differentiate into bone, cartilage, fat cells, cadiocyte, hepatocyte or neuron-like cells. For its profuse resources and easily harvesting, BMSCs have the high values of clinical application.In recent years, many domestic and overseas scholares induced BMSCs to differentiate into neuron-like cells in vitro through various ways. Retinoic acid can induce BMSCs to differentiate into neuron-like cells which can express the earlier neuron marker. The lonely or united utilization of some chemicals such asβ-mercaptoethanol, dimethyl sulfoxide, butylated hydroxyanisole can induce differentiation of BMSCs into neural cells effectively. BNSCs induced by physiological neural differentiation factors also can differentiate into neural cells which have obtained the electrophysiological functions. The hMSCs may have been differentiated into early progenitors of neural cells in vitro under conditions that increase the intracellular level of cAMP. The differentiation rates of BMSCs can be improved by the application of gene transfection techniques or RNAi techniques. Recently, some reseachs teams succeed in inducing the differentiation of BMSCs into neuron-like cells by using chinese crude drug or its simple substance such as: salvia miltiorrhizae, tanshinone A, baicalin, flavol extracting agent, musk polypeptide, Gold Theragran, gastrodia rhizome, berberine etc.. The analysis results indicate that the induction of above-mentioned drugs maybe concerned with the pharmacological action of antioxidation. According to the theory of traditional Chinese medicine, astragalus mongholicus is one of the most important antidotal drugs. The extracting substances of astragalus mongholicus have the pharmacological action of antioxidation and they can remove oxyradical. We guess that the extracting agents of astragalus mongholicus maybe have the capacity of inducing BMSCs to differentiate into neural cells.On account of above-mentioned discussions and in order to establish the foundation for the researches on the curing of ND through stem cellular transplant and the intervention roles of the traditional chinese medicine during the course of cellular transplant, we employ primary cell culture, HE stain, immunocytochemistry, MTT assay, flow cytometry, RT-PCR, etc. to approach the differentiation features of BMSCs induced by astragalus mongholicus at early stage or long-term, cell viability, the changes of the intracellular Ca2+ concentration, the informations of transcription of CaMmRNA. This experiment could be divided into three parts. 1 Bone marrow cells were collected from the femurs and tibia of adult rats on the germ free condition and cultured in L-DMEM supplemented with 15% FBS. Non-adherent cells were eliminated by changing the medium on the next day and the adherent cells were allowed to propagate. BMSCs would be purified by passage. Using invert microscope to observe the growthing condition of BMSCs. Neural differentiation was initiated at passage 4 by using the different concentration of: 40, 100, 200, 400 g/L astragalus mongholicus, control group is free astragalus mongholicus. At 1, 5, 24h after induction, the morphological changes of BMSCs by HE straining were observed with optical microscope. The inducing results were identified by immunocytochemical techniques. The viability of cells was determined by MTT assay. The results showed that the growth-condition of cells was good under the conventional culture condition. BMSCs were fusiform or like fibroblasts. The nucleus of cells was bigger, spherical or elliptical. After induction, the volume of cells was increased, the nucleus of cells was shrunk, long and thin dendrites were formed. Some cells had reticular joint with the concentration of astragalus mongholicus increasing and the induction-time prolonging. The Nestin, NSE, GFAP, and MAP-2 positive straining had been observed during the induction course. The counts of Nestin positive staining cells (126.1±10.63) in the group of 200 g/L-24h were more than other groups significantly (P<0.05). The counts of NSE positive staining cells (135.9±12.17) in the group of 400 g/L -24h were more than other groups significantly (P<0.05). The counts of GFAP positive staining cells were increased with the concentration of astragalus mongholicus increasing and the induction-time prolonging but no significance (P>0.05). The counts of MAP-2 positive staining cells in the group of 200 g/L-24h (25.2±6.66) and 400 g/L-24h (30±6.78) were more than other groups significantly (P<0.05). The results of MTT assay showed that astragalus mongholicus could inhibit the viability of cells in every group at early stage during induction. The inhibition was increased with the concentration of astragalus mongholicus increasing. With the induction went on, astragalus mongholicus could nourish cells and the trophic action was raised with the concentration of astragalus mongholicus increasing. The changing tendency of cell viability was at equal pace between the 200 g/L groups and the 400 g/L groups.2 Basing on the experimental results of the first part, the changes of intracellular Ca2+ concentration ([Ca²⁺]_i) and the informations of transcription of CaM mRNA of BMSCs during the early stage of differentiation induced by 200 g/L astragalus mongholicus were detected to approach the induction mechanisms and signal transduction. The mean fluorescence intensity of Fluo-3-AM (Fn) was measured by flow cytometry before induction and after 10, 30, 60, 120, 300 minutes induction respectively. The informations of transcription of CaM mRNA were detected by RT-PCR before induction and after 30, 60, 120 minutes induction respectively. The results showed that [Ca²⁺]_i changed with induction time. F10 (563±1344) and F30 (511±1250) were more decreased than F0 (677±1584) significantly (P<0.05). F60 (578±1307) rised but was lower than F0 significantly (P<0.05). F120 (845±1648) exceeded F0 significantly (P<0.05). F300 (849±1706) had no significant differences with F120 (P>0.05). The transcription of CaMmRNA changed with induction time. CaM mRNA could be detected in all groups and its relative contents increased gradually. The significant difference existed among every group (P<0.05). 3 Basing on the experimental results of the first part and the second part, the differentiation of BMSCs induced by 200 g/L astragalus mongholicus after 1, 2, 3, 4, 5, 6 day induction were observed by invert microscope and identified by immuocytochemical techniques to expend the application of astragalus mongholicus or its preparation in the course of cellular transplant, which maybe cure ND such as Alzheimer disease and Parkinson disease. The results showed that the morphous of BMSCs changed after induction. The volume of cells increased, the dendrites were formed. As induction went on, the dendrites of cells elongated gradually. The end of dendrites had some branches. The morphous of cells was like neuron. Some cells had reticular joint. After long-term induction, slight cells were suspended in medium. The Nestin positive staining had been observed after 1, 3, 6 day induction. The rates of Nestin positive staining cells after 3 day induction (61.79%) were highest (P<0.05). The rates of NSE positive staining cells after 3 day induction (46.41%) were higher than the rates of NSE positive staining cells after 1 day induction (29.61%)(P<0.05). The rates of NSE positive staining cells after 6 day induction (45.37%) decreased slightly and had no significant difference with the rates after 3 day induction (P>0.05). The GFAP positive staining had been observed after 1, 3, 6 day induction. The rates of GFAP positive staining cells after 3 day induction (52.21%) were higher than the rates of GFAP positive staining cells after 1 day induction (38.77%)(P<0.05). The rates of GFAP positive staining cells after 6 day induction (51.66%) decreased slightly and had no significant difference with the rates after 3 day induction (P>0.05). The rates of MAP-2 positive staining cells after 6 day induction (45.18%) were highest and had significant difference with other groups (P<0.05).Conclusion1 BMSCs can be gained and cultured easily. They can be highly purified by serial subcultivation. They can differentiate into neural stem cells after induction of astragalus mongholicus. On account of the results of immunocytochemical experiments and MTT Assay, we thought that the high-performance induction concentration of astragalus mongholicus is 200 g/L. As times go on, the induction effects have been enhanced.2 [Ca²⁺]_i is down regulation at the early stage of differentiation of BMSCs induced by astragalus mongholicus, but [Ca²⁺]_i is up regulation as induction goes on. With [Ca²⁺]_i being up regulation, the level of CaM mRNA transcription is up regulation meantime. This indicates that the signal transduction channel of Ca2+/CaM maybe play important roles during the differentiation of BMSCs induced by astragalus mongholicus. That [Ca²⁺]_i keeps on the condition of high concentration from 120 min induction to 300 min induction indicates that the neuron-like cells derived from BMSCs after induction maybe have physiological functions of excitable cells.3 At early stage, the induction that astragalus mongholicus induce BMSCs to differentiate into neuron stem cells is high performance and high efficiency. After long-term induction, astragalus mongholicus can induce BMSCs to differentiate into neurons and can induce BMSCs to differentiate into glial cells also. This indicates that the specific effects of astragalus mongholicus after long-term induction are faintness, but we should pay attention to the accelerated growth of dendrite and cell senescence and cell death after long time induction.