【作者】 曾娟；

【导师】 张荣华；

【作者基本信息】 暨南大学 ， 中西医结合临床， 2007， 硕士

【摘要】 目的：观察3月龄SD大鼠MSCs生物学特性，用补肾中药补骨脂的主要活性成分补骨脂素对SD大鼠MSCs进行干预，观察其对体外增殖和成脂分化的影响，确定诱导的最佳添加浓度和添加时间，并通过对诱导MSCs向脂肪细胞分化的相关的重要因子PPARγ的研究，从干细胞的角度探讨补骨脂素影响MSCs分化可能的机制。方法：1．运用全骨髓贴壁法分离培养SD大鼠MSCs，通过流式细胞仪对其表面标志物测定、诱导MSCs向成骨细胞及脂肪细胞分化来鉴定MSCs。2．MTT法检测SD大鼠MSCs的细胞生长曲线；3．将补骨脂素配成5个浓度，用MTT法检测补骨脂素对MSCs增殖的影响，并分别设置对照组；4．将MSCs分为药物+经典诱导组、经典诱导组、空白组，将补骨脂素配成5个浓度，分别用油红O计数法、PNPP法、酶法检测补骨脂素对MSCs脂向分化的影响；5．将MSCs分为药物+经典诱导组、经典诱导组、空白组，运用RT-PCR的方法检测各诱导体系诱导分化过程中与脂肪细胞分化相关重要因子PPARγ的变化。结果：1．MSCs分离后24h基本贴壁，10-15d达到融合，经过成骨、成脂诱导培养，MSCs分别表现出成骨细胞、脂肪细胞表型；2．流式细胞仪检测分离到的大鼠MSCs表面标志抗原CD44、CD29表达阳性，CD45及CD34表达为阴性。3．经MTT法检测补骨脂素对MSCs有增殖作用，发现1μmol／L，5μmol／L的补骨脂素浓度组OD值均高于对照组，P值为别为0.029、0.044(P＜0.05)差别均具有统计学意义。4．采用油红O脂肪细胞计数法，发现5μmol／L、10μmol／L、15μmol／L、20μmol／L浓度的补骨脂素在诱导过程中能够抑制脂肪细胞的产生，P值分别为0.007、0.000、0.000、0.000(P＜0.05)，差别均具有统计学意义。5．经PNPP法与酶法检测，补骨脂素在诱导过程中能够增加ALP的分泌，和抑制TG的分泌。ALP的含量测定：经典诱导组＜药物+经典诱导组＜空白组，P=0.014＜0.05，差别具有统计学意义；TG的含量测定：经典诱导组＞药物+经典诱导组＞空白组，P=0.03＜0.05，差别具有统计学意义；6．补骨脂素对诱导体系中PPARγ表达有下调作用。PPARγ的表达(总体均数)：经典组＞药物+经典诱导组＞空白组(P=0.048＜0.05)，差别具有统计学意义。结论：1．选取3月龄SD大鼠，机械分离，在含10％小牛血清的DMEM-F12培养基中培养可获取较多的MSCs2.1μmol／L和5μmol／L浓度的补骨脂素具有促进MSCs增殖的作用；3.5～20μmol／L浓度的补骨脂素能够抑制MSCs向脂肪细胞分化；降低加入成脂诱导液影响后甘油三酯的含量；提高加入成脂诱导液影响后ALP的分泌4.5μmol／L浓度的补骨脂素能降低MSCs内PPARγmRNA的表达，抑制MSCs的成脂分化；更多还原

【Abstract】 Objective:To observe the biologic characteristic of 3-month SD rats and to observe the effect onproliferation capacity, differentiation to adipocyte of SD rat’s marrow stromal cellcultured by various concentrations of Psoralen and confirm the best concentrationand duration of operation. To investigate the expression of PPARγmRNA in inducedsystem with Psoralen in molecular biology leval. The study may provide a betterunderstanding of the mechanisms involved in the differentiation of 3-month SD rats.Methods:1. To culture MSCs of 3-month SD rats successful. 10% calf serum and DMEM-F12can promote the growth of MSCs in vitro; MSCs from SD rats were purified by usingDifferential Attachment method, then identified according to morphology, FCM andinduced-differentiation potentiality;2. Measuring the cell growth curve of MSCs by means of MTT;3. Different concentrations of Psoralen characterized as 5 concentrations were addedto rat MSCs. Proliferation between Psoralen groups (different concentrations) andblank comparison group were detected by means of MTT;4. Different concentrations of Psoralen characterized as 5 concentrations were addedto rat MSCs which is divided into the Psoralen+induced-differential-to-adipocytegroups (different concentrations), the blank comparison group, and theinduced-differential-to-adipocyte group. To detect the effect on differentiation toadipocyte of SD rat’s MSCs by use of oil-red O staining, PNPP and enzyme method.5.RT-PCR method was used to detect the expression of PPARγ. Grouping was same asabove. Results:1. Primary cultured MSCs adhered to plastic surface within 24 hours and reachedconfluence within 10-15days. Under conditioned inducement, MSCs were successfullydifferentiated into osteoblast, adipocyte;2. MSCs were confirmed that there was expression of CD29 and CD44, and noexpression of CD34 and CD45 on the surface of MSCs.3. The Psoralen have the proliferation effects on MSCs in SD rats. The best promotionof proliferation of MSCs was achieved when the concentration of Psoralen was 1μmol/L and 5μmol/L.(P＜0.05).4. Used oil-red O staining counting method, we found different concentration ofPsoralen such as5μmol/L、10μmol/L、15μmol/L and 20μmol/L in the inductionprocess can inhibit the formation of adipocyte, (P＜0.05), Differences are withstatistical significance.5. We found Psoralen in the induction process has increased the secretion of ALP andinhibit the secretion of TG.. ALP content : induced-differential-to-adipocyte group＜Psoralen+induced-differential-to-adipocyte group＜blank-comparison-group,(P＜0.05); TG content: induced-differential-to-adipocyte group＞Psoralen+induced-differential-to-adipocyte group＞blank-comparison group.6. Psoralen has downward effect on PPARγexpression in induction system. PPARγexpression (overall mean): induced-differential-to-adipocyte group＞Psoraleninduced-differential-to-adipocyte group＞blank-comparison group(P＜0.05).Conclusion:1. MSCs can be cultured and propagated in vitro;2. Some concentrations of Psoralen as 1μmol/L and 5μmol/L have theproliferation effects on MSCs in SD rats.3. Different concentrations of Psoralen characterized as 5～20μmol/L could inhibitadipocyte differentiation, lower TG content, increase the ALP content in MSCs andpromot MSCs into osteoblast.4. 5μmol/L concentration of Psoralen can reduce the expression of PPARγmRNA,更多还原