从调节小肠干细胞功能及其niche研究“益火补土”治法延缓衰老大鼠小肠功能衰退的机制

With the aging of the population trend is growing more and more acute, which is caused by the increase of the age of intestinal digestive and absorptive function attenuation is widespread. Theories of traditional Chinese medicine believe that spleen and intestine are interlinked, according to therapeutic methods of tonifying fire to supplement earth, application of tonifying kidney recipe should be able to improve intestinal function, the modern correlation research has also confirmed that tonifying kidney recipe can significantly protecting intestinal digestion and absorption function of recession, but its mechanism on are still elusive. The intestinal stem cell function decline is the key factor caused of the digestion and absorption function decline, while, stem cell function is determined by its microenvironment (niche), so, whether tonifying kidney recipe can be adjusted through intestinal stem cell niche to postpone intestinal function recession? This project intends to research the disincentive effects of Shenqi decoction on the aging rat intestinal stem cell function of recession as well as in the intestinal stem cell niche of the regulating role, using flow cytometry, Realtime PCR,Western blot and so on, then the antisense RNA technology are utilized Interventions in aging rats the organization of intestinal such as Wnt and Notch signaling etc, and to research effect of Shenqi decoction on intestine stem cell function the regulation of possible molecular signal mechanism, through investigating the effect and mechanism of Tonifying kidney recipe on the protecting intestinal function of recession, to clarify the modern scientific connotation of tonifying fire to supplement earth.

随着人口老龄化日趋严峻，因年龄增加而出现的小肠消化吸收功能衰退普遍存在。中医理论认为，脾与小肠相通，依据“益火补土”治法，补肾方药通过温补命门火应能改善小肠功能，现代相关研究亦证实补肾方药能显著改善衰老所致小肠消化吸收功能衰退，但其作用机制尚不明确。小肠干细胞功能衰退是小肠消化吸收功能衰退的关键因素，而干细胞功能由其微环境（niche）决定，那么，补肾方药能否通过调节小肠干细胞niche进而延缓小肠功能衰退呢？本项目拟先采用流式细胞分析、Realtime PCR、Western blot等技术，研究肾气丸对衰老大鼠小肠干细胞功能衰退的抑制作用及对小肠干细胞niche的调节作用，再采用反义RNA技术干预衰老大鼠小肠组织内Wnt、Notch等信号，研究肾气丸对小肠干细胞功能调节作用的可能分子机制，进而探讨补肾方药延缓衰老所致小肠功能衰退的作用机制，以期阐明“益火补土”治法的现代科学内涵。

干细胞niche变化是导致衰老的根本原因，相关研究均证实，补肾方药具有调节干细胞niche的作用，因而本项目从研究补肾方药对小肠干细胞niche的调节作用为切入点，阐明补肾方药调节小肠干细胞的功能进而延缓小肠功能衰老的机制。结果显示：衰老大鼠十二指肠绒毛长度、绒毛宽度、隐窝深度、V/C值均降低，且PCNA的表达量下降，提示衰老大鼠小肠增殖功能减退。而给予肾气丸后，十二指肠的绒毛长度、绒毛宽度、隐窝深度、V/C值及PCNA的表达量均增加，表明肾气丸可改善衰老大鼠十二指肠的增殖功能。Bmi1、Lgr5、Olfm4是小肠干细胞标记物，研究发现衰老大鼠肠道组织Bmi1、Lgr5、Olfm4表达减少，Wnt/β-catenin 信号通路的相关分子GSK-3β的表达量升高、β-catenin的表达量降低，而给予肾气丸后，小肠Bmi1、Lgr5、Olfm4表达量均升高，β-catenin的表达量也增加，而GSK-3β的表达量降低，提示肾气丸可能通过Wnt/β-catenin信号通路促进小肠干细胞的增殖功能。与成年对照组比较，衰老模型组隐窝杯状细胞数量增多，Notch1、Hes1的表达量降低，给予肾气丸后，与衰老模型组相比，上皮细胞标记物CK8表达减少，隐窝杯状细胞数量减少，杯状细胞分泌的相关蛋白的mRNA（trefoilfactor和mucin3）表达降低，Notch1、Hes1的表达量升高，提示肾气丸可能通过Notch信号通路抑制小肠干细胞向杯状细胞的分化。同时，衰老大鼠十二指肠消化吸收相关酶类ALP、LCT、Pept1基因表达均降低，而给予肾气丸后，ALP、LCT、Pept1基因表达均升高，说明肾气丸可提高衰老大鼠十二指肠的消化吸收功能。此外，我们也研究了另外一种补肾药淫羊藿总黄酮对衰老大鼠肠道干细胞微环境的调节作用，研究显示：给予淫羊藿总黄酮处理的大鼠，与衰老组大鼠相比，空肠隐窝干细胞数量有所增加，空肠增殖分化功能改善，表现为绒毛长度，隐窝深度，V/C值，杯状细胞数量，PCNA表达量增加，且空肠干细胞微环境Wnt/β-catenin和BMP信号与衰老模型组相比呈上调趋势，提示淫羊藿总黄酮也可通过Wnt/β-catenin信号通路促进小肠干细胞的增殖功能。