兔髓核细胞的体外培养及五味子多糖对其生物学行为的影响

发布时间：2019-01-08 16:37

【摘要】：实验目的：应用五味子多糖干预体外培养的兔髓核细胞，通过检测蛋白聚糖和II型胶原的表达情况，观察干预后髓核细胞生物学行为的变化，为椎间盘退变的早期治疗奠定实验基础。实验方法：分离并对兔髓核细胞体外培养，通过免疫组化方法鉴定；当传至第二代，细胞增殖活性达最高时进行干预，实验分为对照组及极低、低、中、高剂量组，采用RT-PCR方法对干预后的细胞进行蛋白聚糖及II型胶原的半定量检测，用TANON GIS凝胶成像处理系统对结果进行半定量分析。MTT法检测细胞增殖情况，绘制生长曲线，观察五味子多糖干预对髓核细胞生物学行为的影响，，可望能延长细胞表型的维持，蛋白聚糖及II型胶原的长期表达。实验结果： 1.体外条件培养兔髓核细胞生长状态良好，活性高；其中以第一、二代为最强，原代活性次之，至第四代细胞活性下降较明显，原代兔髓核细胞4～6d贴壁，指数生长期大约在接种后第16d，大约需22d融合达到90％以上；第一代兔髓核细胞约3左右天贴壁，指数生长期大约在接种后第14d左右，大约需20d达到90％融合。 2. RT-PCR半定量检测结果显示：集聚蛋白聚糖与II型胶原：空白对照组与极低剂量组间无统计学差异，P＞0.05，低、中剂量组与对照组差异明显，有统计学意义，P＜0.05，P＜0.01，且成剂量依赖关系，高剂量组时该作用强度与中剂量实验组相近，无明显的增强作用。 3.MTT法测干预后髓核细胞生长曲线结果显示：实验组髓核细胞增殖速度明显高于对照组。实验结论： 1.体外环境下单层培养法成功培养了兔髓核细胞，经鉴定为髓核细胞。 2.五味子多糖作用于兔髓核细胞后，其II型胶原蛋白及集聚蛋白聚糖表达量增高。且呈现一定的剂量依赖关系。 3.五味子多糖干预兔髓核细胞后，髓核细胞增殖能力显著提高。
[Abstract]:Objective: to investigate the effects of Schisandra chinensis polysaccharides on the expression of proteoglycan and II type collagen in cultured rabbit nucleus pulposus cells. To lay the experimental foundation for early treatment of intervertebral disc degeneration. Methods: rabbit nucleus pulposus cells were isolated and cultured in vitro and identified by immunohistochemistry. When the cell proliferation activity reached the highest level in the second generation, the experiment was divided into control group and very low, medium and high dose groups. The proteoglycan and II type collagen were detected by RT-PCR method. The results were semi-quantitatively analyzed by TANON GIS gel imaging system. MTT method was used to detect cell proliferation, draw growth curve and observe the effect of Schisandra polysaccharide on the biological behavior of nucleus pulposus cells, which could prolong the maintenance of cell phenotype. Long-term expression of proteoglycan and II type collagen. Experimental results: 1. In vitro cultured rabbit nucleus pulposus cells were in good growth state and high activity. Among them, the first and second generation were the strongest, the primary generation activity was the second, and the fourth generation cell activity was obviously decreased. The primary rabbit nucleus pulposus cells adhered to the wall for 4 ~ 6 days, and the exponential growth period was about 16 days after inoculation, and the fusion was more than 90% in 22 days. The first generation of rabbit nucleus pulposus cells adhered to the wall about 3 days after inoculation, the exponential growth period was about 14 days after inoculation, and about 90% fusion was achieved in 20 days. 2. The results of RT-PCR semi-quantitative analysis showed that there was no significant difference between the control group and the very low dose group (P > 0.05), and the difference between the middle dose group and the control group was significant, and the difference between the middle dose group and the control group was significant. P < 0.05 (P < 0.01) and in a dose-dependent manner. The effect intensity of the high dose group was similar to that of the middle dose group, and there was no obvious enhancement effect in the high dose group. The growth curve of nucleus pulposus cells was measured by 3.MTT method. The results showed that the proliferation rate of nucleus pulposus cells in the experimental group was significantly higher than that in the control group. Experimental conclusions: 1. Rabbit nucleus pulposus cells were successfully cultured by monolayer culture in vitro and identified as nucleus pulposus cells. 2. The expression of II type collagen and agglutinin in Schisandra chinensis polysaccharides was increased after it was exposed to rabbit nucleus pulposus cells. There was a dose-dependent relationship. 3. The proliferative ability of Schisandra chinensis polysaccharides in rabbit nucleus pulposus cells was significantly increased.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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