丹参联合hADSCs对脂肪颗粒移植成活率的影响

发布时间：2018-06-28 15:12

本文选题：丹参 + 人脂肪干细胞　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:将丹参与人脂肪来源干细胞联合,通过动物实验,探讨二者综合作用对自体脂肪颗粒植入裸鼠皮下后成活率的影响,进而指导临床应用。方法:体外分离、培养、鉴定hADSCs:脂肪取自接受吸脂手术的1名25岁健康女性的腹部,两次吸脂手术皆是求美者自愿捐赠。取出的脂肪置于无菌离心管中适量的,加入适量磷酸盐缓冲盐水(PBS),运用离心法将脂肪颗粒纯化,去除位于离心管上层的油脂、及位于离心管下层的杂质后,剩余的脂肪颗粒用于hADSCs的制备,第二次抽取的脂肪颗粒同样的方法处理后将脂肪颗粒置于无菌射器中为脂肪颗粒移植备用。选取24只8周龄健康雌性裸鼠,体重(23±3)g,随机分为四组(n=6),A组:脂肪颗粒0.5ml;B组:脂肪颗粒0.5ml+hADSCs;C组:丹参注射液(0.5g·kg~(-1)·d~(-1))[1]+脂肪颗粒0.5ml;D组:丹参注射液(0.5g·kg~(-1)·d~(-1))+脂肪颗粒0.5ml+hADSCs。干细胞与颗粒脂肪比例为:6×105:0.5ml。A、B两组每天腹腔注射与C、D组的丹参注射液等体积的生理盐水。将脂肪颗粒按规定移植入各组裸鼠背部两侧对称的皮下。移植术后15天每组随机选取3只裸鼠处死后取材。取材后,首先对标本进行大体观察,观察组织的体积大小、包膜情况,包膜表面的毛细血管形态特征。然后将标本放入10%甲醛中固定,常规石蜡包埋,切片,将每块标本做5张切片,1张切片用于HE染色,剩余4张切片为免疫组织化学染色备用,用于观察并计数微血管个数。脂肪颗粒移植术后第30天处死各组剩余的裸鼠,将取出的标本进行体积测量。然后将标本用10%甲醛中固定,常规石蜡包埋,切片,每块标本取一张切片做HE染色。结果:1人脂肪干细胞分离、培养及鉴定:细胞提取物由DMEM培养液重悬后,将细胞悬液接种于75cm2的无菌细胞培养瓶中,培养7-9天后培养瓶中可观察到有大量梭形细胞及少量的多角形细胞,多角形细胞几天后会逐渐变形为梭形细胞,14天左右可观察到梭形细胞呈旋涡状或束状生长。由于原代培养脂肪组织中混有少量血管等其他的组织,因此培养瓶中会培养出个别红细胞和其他杂质细胞,而随着脂肪干细胞的传代培养,脂肪干细胞越来越纯。当培养达13~(-1)4天时,原代细胞能达到80%融合,即可进行传代。传代后细胞增长速度加快,每4-5天即可传1代。2流式细胞术检测:检测结果表明CD29、CD44、CD90的表达率均在92%以上;CD31、CD45、CD146阳性率均小于6%,是几乎不表达的,证明该实验中所培养的细胞确实是hADSCs,且h ADSCs属于间充质干细胞的一种。3移植物组织学肉眼观察:移植术后第15天,取出的移植体均可见完整的包膜形成,可见新生毛细血管走形在包膜表面,几组结果对比,新生的毛细血管最为丰富的是D组移植物,新生毛细血管最少的是A组,B,C两组的毛细血管数量介于A、D两组之间;移植术后第30天,移植物的体积D组最大,A组的体积最小,B、C两组的体积大小介于A、D两组体积大小之间。4免疫组化检测移植脂肪的微血管数量:对术后15天各组脂肪移植体免疫组化切片血管密度值采用LSD-t法进行两两比较发现,A组与B组,A组与C组,A组与D组,B组与D组,C组与D组之间的差异具有统计学意义(P0.05),而B组与C组之间的差异无统计学意义(P0.05),可以认为D组的血管密度值最高,A组的血管密度值最低,B组和C组的血管密度值介于A组与D组之间。5丹参联合hADSCs对移植颗粒脂肪体积的影响:术后30天各组脂肪移植体体积值采用LSD-t法进行两两比较发现,A组与B组,A组与C组,A组与D组,B组与D组,C组与D组之间的差异具有统计学意义(P0.05),而B组与C组之间的差异无统计学意义(P0.05),可以认为D组的脂肪移植体体积值最高,A组的脂肪移植体体积值最低,B组和C组的脂肪移植体体积值介于A组与D组之间。结论:丹参与hADSCs的综合作用,比各自单独运用更能有效地促进脂肪颗粒移植体早期血管系统的重建,更有效地提高脂肪颗粒成活率。
[Abstract]:Objective: to combine Salvia miltiorrhiza with human fat derived stem cells and through animal experiments, the effects of the two synthetic effects on the survival rate of autologous fat particles implanted in nude mice were investigated, and then the clinical application was guided. Methods: in vitro isolation, culture, and identification of the abdominal and two liposuction hands of the hADSCs: fat from 1 25 year old women who received liposuction. The fat is placed in the aseptic centrifuge tube. The fat particles are added to the aseptic centrifuge tube, and a proper amount of phosphate buffer salt water (PBS) is added. The fat particles are purified by centrifugation to remove the fat in the upper layer of the centrifuge tube, and after the impurities located in the lower layer of the centrifuge tube, the remaining fat particles are used for the preparation of hADSCs and the second extraction of fat. 24 8 weeks old healthy female nude mice were selected and 24 8 weeks old healthy female nude mice were selected. The body weight (23 + 3) g was randomly divided into four groups (n=6), group A: fat granule 0.5ml; B group: fat granule 0.5ml+hADSCs; C group: Dan Shen Injection (0.5g. Kg~ (-1). D~ (-1)) The ratio of 0.5g / kg~ (-1) / d~ (-1)) + fat particles 0.5ml+hADSCs. stem cells and granular fat was 6 * 105:0.5ml.A, B two was injected into the abdominal cavity every day with C and Salvia miltiorrhiza injection in the D group. The fat particles were transplanted into the subcutaneous symmetry on both sides of the back of the nude mice in each group. 3 nude mice in each group were randomly selected for 15 days after the transplantation. After the rats were sacrificed, the specimens were taken. First, the specimens were observed, the size of the tissue, the envelope, the morphology of the capillaries on the surface of the membrane were observed. Then the specimens were fixed in 10% formaldehyde, the paraffin was embedded and sliced, and 5 slices were made for each specimen, 1 slices were used for HE staining, and the remaining 4 slices were immunized. Thirtieth days after the transplantation of fat particles, the remaining nude mice were killed and the samples were measured. Then the specimens were fixed in 10% formaldehyde, the paraffin was embedded and sliced, and each specimen was sliced for HE staining. Results: 1 human fat stem cells were separated, cultured and evaluated. The cell suspension was suspended by DMEM culture, and the cell suspension was inoculated into the sterile cell culture bottle of 75cm2. A large number of spindle cells and a small number of polygonal cells could be observed in the culture bottle for 7-9 days. The polygonal cells gradually became spindle cells after a few days, and the spindle shaped cells were observed to be vortexed or bundled at about 14 days. As the primary culture adipose tissue is mixed with a small amount of blood vessels and other tissues, a few red cells and other impurity cells will be cultivated in the culture bottle. With the generation of fat stem cells, the fat stem cells are becoming more and more pure. When the culture reaches 13~ (-1) 4 days, the primary cells can reach 80% fusion and can be passaged. The growth rate of the posterior cell was accelerated, and the 1 generation.2 flow cytometry was passed every 4-5 days. The results showed that the expression rate of CD29, CD44 and CD90 was above 92%; CD31, CD45, CD146 positive rates were less than 6%, which were almost not expressed. It proved that the cells cultured in this experiment were hADSCs, and H ADSCs belonged to a kind of.3 graft of mesenchymal stem cells. Histology naked eye observation: fifteenth days after transplantation, all the transplanted bodies were found to form a complete capsule, and the new capillaries were visible on the surface of the capsule. The most abundant new capillaries were group D grafts and the new capillaries were the A group, and the number of capillaries in group B and C two were between A and D two groups. Thirtieth days after transplantation, the size of the D group was the largest, the volume of the A group was the smallest, the volume of the group of B, the two group was between A, and the volume size of the D two groups was measured by.4 immunohistochemistry. The number of the blood vessels of the transplanted fat was detected by the LSD-t method at 15 days after the operation, and the A group and B group, A. The difference between group A and group C, group B and group D, group B and D, C group and D group was statistically significant (P0.05), but the difference between group B and C group was not statistically significant (P0.05). It was considered that the density of the vessel in the D group was the highest and the blood vessel density of the group was the lowest. The effect of the volume of grain fat: 30 days after the operation, the volume value of fat transplantation was compared with the LSD-t method for 22 comparison. The difference between group A and B, group A and C, group A and D, B and D, C group and D group had statistical significance (P0.05), but there was no significant difference between the group and the group. The volume value of the fat graft in group A is the lowest, the volume value of the fat graft in group B and group C is between A and D. Conclusion: the comprehensive effect of Salvia miltiorrhiza and hADSCs is more effective in promoting the reconstruction of the early vascular system of the fat granule transplantation, and more effectively improving the survival rate of fat particles.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R622.9

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