【摘要】：目的:基于衰老相關基因p16~(INK4a)、p53、p21,探討補腎化瘀生新方延緩骨髓間充質干細胞(Bone mesenchymal stem cells,BMSCs)衰老的機制;方法:取60只SPF級SD大鼠,采用隨機數(shù)字表法分為補腎化瘀生新方組和生理鹽水組,用于含藥血清的制備;SPF級SD大鼠,3周齡,進行原代BMSCs的提取,取狀態(tài)良好的第3代BMSCs,用6g/L D-gal誘導建立BMSCs衰老模型;分組:正常組隨機分為空白組、空白中藥組、空白生理鹽水組,衰老組隨機分為模型組、模型中藥組、模型生理鹽水組。觀察各組各代BMSCs的形態(tài)學變化;MTT法確定促進第3代BMSCs分化增殖的最佳含藥血清的濃度;實時熒光定量PCR技術檢測不同代次各組BMSCs p16~(INK4a)、p53、p21 mRNA的表達。結果:(1)鏡下見年輕的BMSCs形態(tài)呈長梭形或紡錘形,漩渦樣分布,生長迅速,折光性好;衰老的BMSCs多呈扁三角形、橢圓形,胞體擴散,生長緩慢,折光性差;(2)根據(jù)MTT法檢測結果可知,促進BMSCs增殖的最佳濃度為10%;(3)實時熒光定量PCR結果顯示:第3代BMSCs,空白組、模型組、模型中藥組、模型生理鹽水組之間相互比較,p16~(INK4a)、p53、p21 mRNA表達量均無統(tǒng)計學意義(P0.05);第6代BMSCs,與空白組相比,模型組、模型生理鹽水組、空白生理鹽水組的三個衰老關鍵基因p16~(INK4a)、p53、p21 mRNA的表達差異具統(tǒng)計學意義(P0.05),空白中藥組、模型中藥組的表達差異無顯著性(P0.05);與模型組相比,模型中藥組的表達差異具有顯著性統(tǒng)計學意義(P0.01),模型生理鹽水組的表達無統(tǒng)計學意義(P0.05);且模型中藥組與模型生理鹽水組、空白中藥組與空白生理鹽水組相比,p16~(INK4a)、p53、p21 mRNA的表達差異均具有統(tǒng)計學意義(P0.05)。第7代BMSCs,與空白組相比,空白中藥組p16~(INK4a)、p53、p21 mRNA的表達差異無統(tǒng)計學意義(P0.05),余四個組的表達差異均具統(tǒng)計學意義(P0.05);與模型組相比,模型中藥組的三個基因的表達具有統(tǒng)計學意義(P0.05),模型生理鹽水組的表達無統(tǒng)計學意義(P0.05);模型中藥組與模型生理鹽水組、空白中藥組與空白生理鹽水組相比,差異均具有統(tǒng)計學意義(P0.05)。第9代BMSCs,與空白組相比,空白中藥組p16~(INK4a)、p53、p21 mRNA的表達量差異無統(tǒng)計學意義(P0.05),余四個組的表達量差異均具有顯著性(P0.01);與模型組相比,模型中藥組p16~(INK4a) mRNA的表達量無統(tǒng)計學意義(P0.05),p53、p21 mRNA的表達量差異有統(tǒng)計學意義(P0.05),而模型生理鹽水組三個基因mRNA的表達量差異均無統(tǒng)計學意義(P0.05);與空白中藥組相比,空白生理鹽水組三個基因mRNA的表達均具有統(tǒng)計學意義(P0.05);與模型中藥組相比,模型生理鹽水組p16~(INK4a) mRNA的表達不具有統(tǒng)計學意義(P0.05),模型生理鹽水組p53、p21 mRNA的表達量差異均具有統(tǒng)計學意義(P0.05)。結論:(1)在自然衰老及成功誘導衰老模型的條件下,補腎化瘀生新方可延緩BMSCs的衰老,但對年輕的BMSCs作用不明顯;(2)補腎化瘀生新方可通過下調衰老關鍵基因p16~(INK4a)、p53、p21的表達來延緩BMSCs的衰老,但隨著衰老時間的延長,其作用力逐漸減弱,并且到一定程度,這種衰老具有不可逆性;(3)補腎化瘀生新方調控BMSCs的衰老與其抑制p16/pRb和p53/p21途徑密切相關。
[Abstract]:Objective: To investigate the mechanism of delaying aging of bone marrow mesenchymal stem cells (BMSCs) based on senescence-related genes p16 ~ (INK4a), p53 and p21. Methods: 60 SPF SD rats were divided into two groups: Bushen Huayu Xinfang group and normal saline group. The method comprises the following steps of: preparing a drug-containing serum; SPF-grade SD rats; 3-week age; carrying out extraction of primary BMSCs; taking the third generation BMSCs with good condition; inducing the establishment of BMSCs aging model by using 6g/ L D-retinoic acid; grouping: the normal group is randomly divided into a blank group, a blank Chinese medicine group and a blank physiological saline group; The aging group was randomly divided into model group, model Chinese medicine group and model physiological saline group. Morphological changes of BMSCs were observed in each group; MTT method was used to determine the concentration of the best drug-containing serum for the differentiation and proliferation of the third generation BMSCs; and the expression of p16 ~ (INK4a), p53 and p21 mRNA in different generations of BMSCs was detected by real-time fluorescence quantitative polymerase chain reaction (PCR). Results: (1) The morphology of the young BMSCs under the scope of the mirror was in the form of a long shuttle shape or a crescent shape, the spiral pattern was distributed, the growth was rapid, the refractive index was good; the aging BMSCs were flat triangles, oval, cell diffusion, slow growth and poor refractive index; (2) According to the MTT method, it can be seen that, The optimal concentration of BMSCs was 10%; (3) Real-time fluorescence quantitative PCR showed that the expression of p16 ~ (INK4a), p53 and p21 mRNA was not statistically significant (P0.05). Compared with the blank group, the expression of p16 ~ (INK4a), p53 and p21 mRNA in the model group, normal saline group and blank saline group was statistically significant (P0.05). There was no significant difference in the expression of the model Chinese medicine group (P0.01), the expression of the model physiological saline group was not statistically significant (P0.05), and compared with the blank normal saline group, p16 ~ (INK4a) and p53 were found in the model Chinese medicine group. The expression of p21 mRNA was statistically significant (P0.05). Compared with the blank group, the expression of p16 ~ (INK4a), p53 and p21 mRNA in the blank Chinese medicine group was not statistically significant (P0.05). There was no significant difference in the expression of normal saline group (P0.05). There was no significant difference in the expression of p16 ~ (INK4a), p53 and p21 mRNA in the blank Chinese medicine group compared with the blank group (P <0.05). The expression of p16 ~ (INK4a) mRNA in the model Chinese medicine group was not statistically significant (P0.05). There was no significant difference in the expression of p21 mRNA in normal saline group (P0.05). Compared with the traditional Chinese medicine group, the expression of p16 ~ (INK4a) mRNA in normal saline group was not statistically significant (P0.05). Conclusion: (1) Under the condition of natural aging and successful induction of aging model, the aging of BMSCs can be delayed by tonifying kidney and blood stasis, but it is not obvious to young BMSCs; (2) BMSCs can be delayed by lowering the expression of p16 ~ (INK4a), p53 and p21. However, with the prolongation of aging time, its acting force is gradually weakened, and to some extent, this kind of aging has irreversibility; (3) The aging of BMSCs is closely related to the inhibition of p16/ pRb and p53/ p21.
【學位授予單位】：河南中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R285.5

【相似文獻】

相關期刊論文 前10條

1 姚淑敏;衰老分子生物學研究進展[J];華北煤炭醫(yī)學院學報;2001年02期

2 陳國華,潘光輝;中醫(yī)藥延緩衰老的現(xiàn)代研究概況[J];濰坊醫(yī)學院學報;2004年06期

3 肖政華;張光奇;;衰老的分子基因水平研究[J];貴陽中醫(yī)學院學報;2006年03期

4 楊發(fā)亮;房冬梅;鄒銀平;;淺析運動延緩衰老的機制[J];內江科技;2011年10期

5 王福龍;王甄真;陳雁;;衰老的分子機制與干預研究的最新進展[J];中國細胞生物學學報;2012年08期

6 孫明德;鄭集;;衰老的起因和機制[J];生理科學進展;1981年02期

7 吳鏡海;;人類生理衰老生物學[J];海南大學學報(自然科學版);1985年03期

8 Roy A K;汪義和;;各種衰老理論的評價[J];國外醫(yī)學(老年醫(yī)學分冊);1986年04期

9 南新升,張宗玉,童坦君,張昌穎;衰老的遺傳控制[J];老年學雜志;1990年02期

10 陳禮湘;老年生理(1)[J];護士進修雜志;1992年01期

相關會議論文 前10條

1 朱漢民;;衰老的生物指標[A];中國營養(yǎng)學會第三屆老年營養(yǎng)暨第二屆營養(yǎng)與腫瘤學術會議論文摘要匯編[C];1994年

2 傅文慶;;衰老及其治療[A];老齡問題研究論文集（七）[C];2005年

3 童坦君;張宗玉;王文恭;;衰老相關基因研究進展[A];老齡問題研究論文集（十一）——積極老齡化研究之三[C];2006年

4 任利群;;衰老與心血管疾病[A];第十屆全國老年醫(yī)學進展學術會議暨江蘇省中西醫(yī)結合學會老年分會學術交流會論文集[C];2010年

5 姚軍孝;毛忠南;王建文;汪海燕;毛立亞;張曉凌;張恩育;;中醫(yī)對衰老的認識和中醫(yī)藥抗衰老機制研究[A];甘肅省中醫(yī)藥學會2013年學術年會論文集[C];2013年

6 彭小冬;張宗玉;童坦君;;衰老相關p21~(WAF1)的高表達并非與p53無關[A];中國生物化學與分子生物學會第八屆會員代表大會暨全國學術會議論文摘要集[C];2001年

7 姚軍孝;毛忠南;王建文;汪海燕;毛立亞;張曉凌;張恩育;;中醫(yī)對衰老的認識和中醫(yī)藥抗衰老機制研究[A];全國針灸臨床適宜技術推廣研討會暨甘肅省針灸學會2013年學術年會論文集[C];2013年

8 姜明紅;項洋;張亞璽;劉士廉;劉彥信;鄭德先;;miRNA在免疫衰老中的調控機制研究[A];遺傳學與社會可持續(xù)發(fā)展——2010中國青年遺傳學家論壇論文摘要匯編[C];2010年

9 呂曉鳳;蔣曉剛;白俊海;毛澤斌;;IGFBP-3基因在衰老細胞中的表達調控研究[A];第七屆全國老年醫(yī)學學術會議暨海內外華人老年醫(yī)學學術會議論文匯編[C];2004年

10 謝肖立;吳s，

本文編號：2297466