【摘要】 目的:收集体外培养、纯化的骨髓间质干细胞条件培养液,检测其对多巴胺能神经元有保护作用的胶质源性神经生长因子分泌情况,并观察对1-甲基4-苯基吡啶离子诱导的PC12细胞凋亡的保护作用。方法:实验于2005-05/2006-10在中国医科大学附属一院神经内科实验室完成。①PC12细胞由协和医科大学细胞培养中心提供。1-甲基4-苯基吡啶离子(Sigma,USA,批号3707312)。②选取清洁级SD大鼠20只,麻醉后取股骨和胫骨,去净肌肉取骨髓,按1010L-1接种于含体积分数为0.2胎牛血清的低糖型DMEM培养基中,通过弃悬浮细胞及换液后可得到较纯的骨髓间质干细胞。培养第10天胰蛋白酶消化传代,当第2代细胞扩增至铺满瓶底80%时,改用含体积分数为0.05胎牛血清的低糖型DMEM条件培养液,48h后收集培养液,经超滤浓缩系统(截留分子量为10000)浓缩10倍后过滤除菌。③骨髓间充质干细胞接种于24孔板内,贴壁后多聚甲醛固定,磷酸盐缓冲液漂洗,免疫荧光法鉴定骨髓间充质干细胞表面抗原的表达。骨髓间充质干细胞消化后进行细胞计数,按1×108L-1接种于75cm培养瓶,加入含体积分数为0.2胎牛血清的DMEM培养液20mL,于培养第5,10天采用ELISA法测定细胞培养上清液中的胶质源性神经生长因子含量。④PC12细胞置于RPMI1640培养液中,内含体积分数为0.1的马血清和0.05的胎牛血清,汇集至80%时进行传代接种,液氮罐中贮存备用。实验前首先置换培养液,使血清浓度降至为仅含0.01马血清和0.01胎牛血清,24h后将培养的细胞分为4组:空白对照组,在细胞培养体系中不加入任何药物;骨髓间充质干细胞上清液组,接种后24h在细胞培养体系中分别加入骨髓间充质干细胞上清液30,60,120μL;1-甲基4-苯基吡啶离子组,接种后24h分别加入1-甲基4-苯基吡啶离子,使终浓度分别为100,200,400μmol/L;联合组,接种后24h加入200μmol/L1-甲基4-苯基吡啶离子,24h后再分别给予骨髓间充质干细胞上清液30,60,120μL。⑤各组细胞于给药后24,48h,流式细胞术检测PC12细胞的凋亡率;通过免疫细胞化学法和RT-PCR法检测PC12细胞半胱氨酸天冬氨酸蛋白酶3蛋白和mRNA的表达。结果:①骨髓间充质干细胞表面抗原鉴定:骨髓间充质干细胞可在体外分离扩增,其表面抗原CD45呈阴性,而CD44表达阳性。②骨髓间充质干细胞培养上清液中胶质源性神经生长因子水平检测:第5,10天培养上清液中的胶质源性神经生长因子浓度为(44.57±5.96)ng/L和(45.41±6.33)ng/L,差异无显著性意义(P>0.05)。③PC12细胞凋亡情况:联合组200μmol/L1-甲基4-苯基吡啶离子作用PC12细胞24h后,细胞凋亡率为(42.34±3.21)%;加入30,60,120μL骨髓间充质干细胞上清液处理24h后,细胞凋亡率分别降为(31.96±2.89)%,(17.89±1.78)%,(10.08±0.91)%,差异有显著性意义(P<0.05)。联合组药物作用48h与24h情况相似。④给药后各组半胱氨酸天冬氨酸蛋白酶3蛋白及mRNA的表达:联合组半胱氨酸天冬氨酸蛋白酶3蛋白和mRNA水平均明显低于1-甲基4-苯基吡啶离子组(t=0.05～0.32,P均<0.05)。结论:骨髓间充质干细胞能够分泌胶质源性神经生长因子,对1-甲基4-苯基吡啶离子诱导的PC12细胞凋亡产生保护作用。这种保护作用的强弱与其浓度有关,具体作用机制可能是通过抑制半胱氨酸天冬氨酸蛋白酶3蛋白和mRNA水平实现的。更多还原

【Abstract】 AIM: The culture supernatant of the mesenchymal stem cells (MSCs) cultured and purified in vitro were collected to detect secretary instance of the glial cell line-derived neurotrophic factors which have the protective function to the dopaminergic neurons. To observe the protective effect of MSCs secretary factors on 1-methyl-4-phenylpyridine ion (MPP+) induced apoptosis in pheochromocytoma (PC12) cells. METHODS: The experiment was finished in Lab of Department of Neurology , First Affiliated Hospital of China Medical University from May 2005 to October 2006. ①PC12 cells were offered by Cell Culture Center of Union Medical University. There was MPP+ (Sigma, USA, lot number 3707312). ②20 pureness grade SD rats were selected, taken thighbone and tibia after anaesthesia, got marrow after scraping off all attached muscles and the marrow was plated in culture flask according to 1010 L-1 in low glucose Dulbecco’s modified Eagle’s medium (DMEM), containing 0.2 fetal bovine serum and pure MSCs were gained by abandoning suspending cells and exchanged fluid. They were digested and passed generation with trypsin in the culturing tenth day. Low glucose DMEM, containing 0.05 fetal bovine serum, was used when the second generation cells were plated in 80% culture flask bottom. The culture supernatant was collected after 48 hours, filtrated and removed bacteria by filter concentration system (sectional molecular weight was 10 000) concentrating 10 times. ③MSCs were inoculated in 24 aperture plank, fixed with polyformaldehyde after adhering the wall, washed by phosphate solution and surface antigen of MSCs was measured by immunocytochemistry. MSCs were counted after digesting, inoculated in 75 cm culture flask according to 1×108 L-1 in 20 mL low glucose DMEM containing 0.2 fetal bovine serum and contents of glial cell line-derived neurotrophic factor(GDNF)in the culture supernatant were detected by ELISA in the culturing fifth and tenth days. ④PC12 cells were placed in RPMI1640 culture solution, containing 0.05 fetal bovine serum and 0.1 Equine serum, passed generation when the cells were collected in 80% culture flask and reserved in liquid nitrogen jar. Culture solution was replaced and decreased serum concentration to 0.01 fetal bovine serum and 0.01 Equine serum. PC12 were divided into 4 groups after 24 hours: blank control group. It was not joined any medicine in the cell culture system; simple supernatant of MSCs group, it was joined the supernatant of MSCs in the cell culture system after inoculating 24 hours, the concentration was 30,60,120 μL; simple MPP+ group, it was joined the MPP+ in the cell culture system after inoculating 24 hours and eventual concentration was 100,200,400 μmol/L, respectively; combination group, it was joined the 30,60,120 μL supernatant of MSCs after 24 hours following joining the 200 μmol/L MPP+. ⑤Apoptotic rate was analyzed by flow Cytometric (FCM). The expressions of the protein and mRNA of caspase 3 were measured by immunocytochemistry and RT-PCR techniques, respectively after joined medicine 24 hours and 48 hours. RESULTS: ①MSCs surface antigen measurement: MSCs could be isolated and proliferated in vitro, and it was positive for CD44 and negative for CD45. ②measurement of the levels of GDNF in the culture supernatant: The concentrations in the culturing fifth and tenth day of GDNF were (44.57±5.96) ng/L and (45.41±6.33) ng/L, and there was no significant difference (P > 0.05). ③apoptosis of PC12 cells: The apoptotic rate of combination group 24 hours following joining the 200 μmol/L MPP+ was (42.34±3.21)%. The apoptotic rate decreased to (31.96±2.89)%, (17.89±1.78)%, (10.08±0.91)%, respectively 24 hours after 30,60,120 μL supernatant of MSCs, and there were significant difference (P < 0.05). The drug effect was similar at hours 48 and 24 in the combination group. ④The expressions of protein and mRNA of caspase 3 in every group: The levels of protein and mRNA of caspase 3 in combination group were obviously lower than those in the simple MPP+ group (t =0.05-0.32,P < 0.05). CONCLUSION: MSCs can secrete GDNF and have protective effect against MPP+ induced apoptosis in PC12 cells. Its strong and weak was connected with different concentrations. Its inhibiting apoptosis mechanism may be achieved by deregulating the levels of protein and mRNA of caspase 3.更多还原