【摘要】：目的：研究三七總皂苷(tPNS)與堿性成纖維細胞生長因子(bFGF)在人骨髓間充質干細胞(hBMSCs)分化為神經(jīng)元樣細胞過程中的誘導、抗凋亡作用及作用劑量。 方法：hBMSCs取自健康成年志愿者，全骨髓貼壁法分離、培養(yǎng)、擴增、純化hBMSCs，流式細胞儀檢測hBMSCs表面的CD29、CD44、CD105、CD34分子，并測定細胞周期。取第3代hBMSCs，含20%胎牛血清的1mmol/Lβ-巰基乙醇(β-ME)預誘導24h，繼而用20ng/mLbFGF（標準對照組）、1.0mg/mL tPNS（藥物組）、20ng/mL bFGF+(0.5 mg/mL、1.0 mg/mL、2.0 mg/mL) tPNS（低、中、高劑量聯(lián)合組）誘導液誘導，設立空白對照組。采用免疫細胞化學法鑒定神經(jīng)細胞特異性抗原標記神經(jīng)元特異性烯醇化酶(NSE)、微管相關蛋-2(MAP-2)和神經(jīng)膠質纖維酸性蛋白(GFAP)的表達；MTT法檢測各組誘導劑不同時間點對神經(jīng)元樣細胞活力的影響，TUNEL、Annexin V-FITC/PI凋亡試劑盒測定誘導后各組細胞凋亡率。 結果: 1、成功分離培養(yǎng)出hBMSCs，流式細胞儀檢測表面標記為：CD29(+)、CD44(+)、CD105(+)、CD34(-)，分離培養(yǎng)的細胞符合hBMSCs表型特征；測定細胞周期示：高比例的G0/G1期細胞提示hBMSCs具有高度分化潛能； 2、免疫細胞化學法測定誘導后細胞：聯(lián)合組NSE、MAP-2陽性細胞比例高于空白對照組、標準對照組及藥物組(P0.05)，，且bFGF與藥物聯(lián)合組各組之間隨著藥物劑量的增加神經(jīng)元樣細胞特異性標志物陽性率也增加(P0.05)，GFAP表達陰性； 3、MTT測定誘導后細胞活力示：聯(lián)合組細胞的活性較對照組高(P0.05)，聯(lián)合組間隨著藥物劑量的增加細胞活性也增高(P0.05)，且細胞活性高、存活時間延長。 4、分別用TUNEL、Annexin V-FITC/PI凋亡試劑盒檢測誘導后各組細胞凋亡率，誘導后聯(lián)合組細胞凋亡率明顯下降(P0.05)，且聯(lián)合組中隨著藥物劑量的增加凋亡率隨之下降(P0.05)。 結論：1、tPNS與bFGF均可誘導hBMSCs分化為神經(jīng)元樣細胞，聯(lián)合組更能有效誘導使其表達神經(jīng)細胞表面特異性抗原，且細胞活力較好，與藥物劑量呈正相關； 2、tPNS與bFGF誘導hBMSCs分化為神經(jīng)元樣細胞后增殖能力強，聯(lián)合組誘導后細胞增殖能力及活力更強，存活時間明顯延長，與藥物劑量呈正相關； 3、tPNS與bFGF誘導hBMSCs分化為神經(jīng)元樣細胞后凋亡率下降，tPNS與bFGF聯(lián)合誘導可明顯減少神經(jīng)元樣細胞凋亡率，與藥物劑量呈負相關。
[Abstract]:Aim: to study the induction of panax notoginseng saponins (tPNS) and basic fibroblast growth factor (bFGF) in differentiation of human bone marrow mesenchymal stem cells (hBMSCs) into neuron-like cells. Methods: human hBMSCs were isolated from healthy adult volunteers, isolated, cultured, amplified and purified by whole bone marrow adherent method. Flow cytometry was used to detect CD29, CD44, CD105 and CD34 molecules on the surface of hBMSCs, and the cell cycle was measured. 1mmol/L 尾 -mercaptoethanol (尾 -ME) containing 20% fetal bovine serum was taken from the third generation hBMSCs for 24 hours, and then was induced by 1.0 mg / mL tPNS (drug group) with 20ng/mLbFGF (standard control group) and 20 mg / mL bFGF (0.5 mg / mL mLX 1.0 mg / mL 2.0 mg/mL) tPNS (). The expression of neuron-specific enolase (NSE), microtubule-associated egg -2 (MAP-2) and glial fibrillary acidic protein (GFAP) was detected by immunocytochemistry. The effect of inducer on neuron-like cell viability was detected by MTT assay. Apoptosis rate of each group was determined by Tunel Annexin V-FITC/PI assay. Results: 1. HBMSCs were isolated and cultured successfully, and the surface markers were detected by flow cytometry as: 1) CD29 () CD44 () CD44 () / CD105 () / CD34 (-). The results of cell cycle analysis showed that the high proportion of G0/G1 phase cells indicated that hBMSCs had high differentiation potential. 2.Immunocytochemistry method was used to determine the percentage of NSEP MAP-2 positive cells in the combined group than in the blank control group. The positive rate of neuron-like cell specific markers in the standard control group and drug group (P0.05) was also increased with the increase of drug dose (P0.05). 3MTT assay showed that the cell activity of the combined group was higher than that of the control group (P0.05), and the activity of the combined group was also increased with the increase of the drug dose (P0.05), and the cell activity of the combined group was higher than that of the control group (P0.05). The survival time was prolonged. 4. The apoptosis rate of each group was detected by Tunel Annexin V-FITC/PI apoptosis kit after induction. After induction, the apoptosis rate of combined group decreased significantly (P0.05), and the apoptosis rate decreased with the increase of drug dosage (P0.05). Conclusion both hBMSCs and bFGF can induce the differentiation of hBMSCs into neuron-like cells. The combined group can effectively induce the expression of neuronal surface specific antigen, and the cell viability is better, which is positively correlated with the dosage of the drug. 2the proliferation ability of hBMSCs differentiated into neuron-like cells induced by TPN and bFGF was stronger than that of the combined group, and the survival time was significantly prolonged, which was positively correlated with the dosage of drug. 3The apoptosis rate of hBMSCs differentiated into neuron-like cells induced by bFGF and tPNS was decreased. The apoptosis rate of neuron-like cells induced by tPNS and bFGF was significantly decreased, which was negatively correlated with drug dose.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

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