【摘要】：實(shí)驗(yàn)?zāi)康模簯?yīng)用五味子多糖干預(yù)體外培養(yǎng)的兔髓核細(xì)胞，通過(guò)檢測(cè)蛋白聚糖和II型膠原的表達(dá)情況，觀察干預(yù)后髓核細(xì)胞生物學(xué)行為的變化，為椎間盤退變的早期治療奠定實(shí)驗(yàn)基礎(chǔ)。 實(shí)驗(yàn)方法：分離并對(duì)兔髓核細(xì)胞體外培養(yǎng)，通過(guò)免疫組化方法鑒定；當(dāng)傳至第二代，細(xì)胞增殖活性達(dá)最高時(shí)進(jìn)行干預(yù)，實(shí)驗(yàn)分為對(duì)照組及極低、低、中、高劑量組，采用RT-PCR方法對(duì)干預(yù)后的細(xì)胞進(jìn)行蛋白聚糖及II型膠原的半定量檢測(cè)，用TANON GIS凝膠成像處理系統(tǒng)對(duì)結(jié)果進(jìn)行半定量分析。MTT法檢測(cè)細(xì)胞增殖情況，繪制生長(zhǎng)曲線，觀察五味子多糖干預(yù)對(duì)髓核細(xì)胞生物學(xué)行為的影響，，可望能延長(zhǎng)細(xì)胞表型的維持，蛋白聚糖及II型膠原的長(zhǎng)期表達(dá)。 實(shí)驗(yàn)結(jié)果： 1.體外條件培養(yǎng)兔髓核細(xì)胞生長(zhǎng)狀態(tài)良好，活性高；其中以第一、二代為最強(qiáng)，原代活性次之，至第四代細(xì)胞活性下降較明顯，原代兔髓核細(xì)胞4～6d貼壁，指數(shù)生長(zhǎng)期大約在接種后第16d，大約需22d融合達(dá)到90％以上；第一代兔髓核細(xì)胞約3左右天貼壁，指數(shù)生長(zhǎng)期大約在接種后第14d左右，大約需20d達(dá)到90％融合。 2. RT-PCR半定量檢測(cè)結(jié)果顯示：集聚蛋白聚糖與II型膠原：空白對(duì)照組與極低劑量組間無(wú)統(tǒng)計(jì)學(xué)差異，P＞0.05，低、中劑量組與對(duì)照組差異明顯，有統(tǒng)計(jì)學(xué)意義，P＜0.05，P＜0.01，且成劑量依賴關(guān)系，高劑量組時(shí)該作用強(qiáng)度與中劑量實(shí)驗(yàn)組相近，無(wú)明顯的增強(qiáng)作用。 3.MTT法測(cè)干預(yù)后髓核細(xì)胞生長(zhǎng)曲線結(jié)果顯示：實(shí)驗(yàn)組髓核細(xì)胞增殖速度明顯高于對(duì)照組。 實(shí)驗(yàn)結(jié)論： 1.體外環(huán)境下單層培養(yǎng)法成功培養(yǎng)了兔髓核細(xì)胞，經(jīng)鑒定為髓核細(xì)胞。 2.五味子多糖作用于兔髓核細(xì)胞后，其II型膠原蛋白及集聚蛋白聚糖表達(dá)量增高。且呈現(xiàn)一定的劑量依賴關(guān)系。 3.五味子多糖干預(yù)兔髓核細(xì)胞后，髓核細(xì)胞增殖能力顯著提高。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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