本文選題：γ氨基丁酸　切入點(diǎn)：腸干細(xì)胞　出處：《山東大學(xué)》2016年碩士論文

【摘要】：研究目的：癌癥研究已經(jīng)在預(yù)防、早期診斷以及尋找惡性腫瘤特定的分子標(biāo)記上獲得了突出的成果。然而,如何防止因?yàn)槭褂没熓侄沃委熗砥谀[瘤轉(zhuǎn)移所引起的正常組織受損仍然有著很大的挑戰(zhàn)。γ氨基丁酸可以抑制神經(jīng)干細(xì)胞增殖,而本研究發(fā)現(xiàn)腸上皮表達(dá)γ氨基丁酸能信號(hào)系統(tǒng),并且丫氨基丁酸參與調(diào)節(jié)腸上皮干細(xì)胞的增殖與分化。于是,我們嘗試檢測(cè)是否通過(guò)藥物干預(yù)γ氨基丁酸能信號(hào)系統(tǒng)可以促進(jìn)腸干細(xì)胞增殖來(lái)加速腸上皮的由于化療導(dǎo)致的損傷修復(fù)。研究方法：利用免疫組織化學(xué)技術(shù)確認(rèn)γ氨基丁酸A型及B型受體亞基、合成酶、轉(zhuǎn)運(yùn)體和降解酶在小鼠及人腸上皮的表達(dá)分布；給予成年C57小鼠γ氨基丁酸A型受體激動(dòng)劑muscimol、拮抗劑bicuculline、B型受體激動(dòng)劑baclofen及拮抗劑CGP54626腹腔注射2周,取空腸組織做HE染色觀察腸形態(tài),免疫組化染色分析比較暫時(shí)性增殖細(xì)胞數(shù)目、BrdU+細(xì)胞數(shù)目、杯狀細(xì)胞數(shù)目及凋亡細(xì)胞數(shù)目；給予成年C57小鼠連續(xù)5天治療劑量或單次大劑量5氟尿嘧啶腹腔注射建立腸損傷模型,同時(shí)注射bicuculline或者CGP54626,取空腸組織做HE染色觀察腸形態(tài),免疫組化染色分析比較暫時(shí)性增殖細(xì)胞數(shù)目、BrdU+細(xì)胞數(shù)目、杯狀細(xì)胞數(shù)目、凋亡細(xì)胞數(shù)目及γH2AX+細(xì)胞；利用免疫組化染色β-catenin探究Wnt信號(hào)通路,使用QPCR技術(shù)檢測(cè)常備干細(xì)胞標(biāo)志物lgr5、 olfm4與儲(chǔ)備干細(xì)胞標(biāo)志物]mTert、bmi mRNA相對(duì)表達(dá)量以及DNA損傷相關(guān)基因的mRNA相對(duì)表達(dá)量。研究結(jié)果：1. GAD65、GAD67、GAT3、GABAARa1、β1/2/3、GABABRI、 GABABR2在腸上皮都有表達(dá),且在隱窩表達(dá)較多,此外GABAARπ、α5、δ γ2、GAT2均在腸上皮表達(dá),而ABAT幾乎不在腸上皮表達(dá)。2.腹腔注射muscimol減少了絨毛長(zhǎng)度、暫時(shí)性增殖細(xì)胞、杯狀細(xì)胞的數(shù)目,增加了凋亡細(xì)胞的數(shù)目；bicuculline增加了絨毛長(zhǎng)度、杯狀細(xì)胞、潘氏細(xì)胞數(shù)目;baclofen減少了暫時(shí)性增殖細(xì)胞數(shù)目而CGP54626則增加了暫時(shí)性增殖細(xì)胞數(shù)目。3.在化療藥物誘導(dǎo)的腸損傷模型中腹腔注射bicuculline或CGP54626相較生理鹽水減少了絨毛受損,增加了暫時(shí)性增殖細(xì)胞數(shù)目,減少了γH2AX+細(xì)胞的數(shù)目,olfm4、lgr5的mRNA相對(duì)表達(dá)量較高,而bmi1、mTert的mRNA相對(duì)表達(dá)量較低,注射CGP54646同時(shí)減少了干細(xì)胞的凋亡細(xì)胞數(shù)目,且gtf2h2與prkdc的mRNA表達(dá)量下降。結(jié)論：1.γ氨基丁酸能信號(hào)系統(tǒng)存在于腸道上皮。2.γ氨基丁酸通過(guò)其A型受體參與調(diào)控腸上皮細(xì)胞增殖、凋亡與分化。3.γ氨基丁酸通過(guò)其B型受體參與調(diào)控腸上皮細(xì)胞增殖。4.抑制γ氨基丁酸A型受體或者B型受體可以保護(hù)化療藥物導(dǎo)致的腸損傷。
[Abstract]:Research objective: cancer research has achieved significant results in prevention, early diagnosis and the search for specific molecular markers for malignant tumours. However, How to prevent normal tissue damage caused by chemotherapy in the treatment of advanced tumor metastasis remains a challenge. GABA can inhibit the proliferation of neural stem cells. In this study, we found that intestinal epithelium expressed gamma-aminobutyric acid signaling system, and ABA was involved in regulating the proliferation and differentiation of intestinal epithelial stem cells. We try to detect whether drug intervention in the gamma-aminobutyric acid signaling system can promote the proliferation of intestinal stem cells to accelerate chemotherapy-induced damage repair of intestinal epithelium. Gamma aminobutyric acid type A and B receptor subunits, Expression and distribution of synthetase, transporter and degradase in mouse and human intestinal epithelium, muscimol, an agonist of gamma-aminobutyric acid type A receptor in adult C57 mice, baclofen, an antagonist, and CGP54626, an antagonist, were injected intraperitoneally for 2 weeks. Jejunum tissues were stained with HE to observe the shape of intestine. The number of temporary proliferative cells and the number of BrdU cells, goblet cells and apoptotic cells were analyzed by immunohistochemistry. Adult C57 mice were given intraperitoneal injection of 5-fluorouracil for 5 days or a single dose of 5-fluorouracil to establish a model of intestinal injury. Jejunum tissues were taken for HE staining to observe the shape of the intestine, and bicuculline or CGP54626 were injected simultaneously. The number of BrdU cells, goblet cells, apoptotic cells and 緯 H2AX cells were analyzed by immunohistochemical staining. 尾 -catenin immunohistochemical staining was used to explore the Wnt signaling pathway. QPCR technique was used to detect the relative expression of mTertnbmi mRNA and DNA damage related gene by QPCR. The results were as follows: 1. GAD65GAD67GAD67GAD67GAD67GABAARA 1, 尾 1 / 2 / 3 GABABRI, GABABR2 were expressed in intestinal epithelium, and expressed more in crypt. In addition, GABAAR 蟺, 偽 5, 未 緯 2GAT2 were expressed in intestinal epithelium, while ABAT was hardly expressed in intestinal epithelium. Intraperitoneal injection of muscimol reduced villi length, temporary proliferative cells, goblet cells, increased the number of apoptotic cells and increased the length of villi and goblet cells. In the model of intestinal injury induced by chemotherapeutic drugs, bicuculline or CGP54626 were injected intraperitoneally to reduce villi damage, but CGP54626 increased the number of transient proliferating cells. It increased the number of temporary proliferative cells and decreased the number of 緯 H2AX cells. The relative expression of mRNA was higher in 緯 H2AX cells, but the relative expression of mRNA in bmi1mTert was lower. CGP54646 injection also decreased the number of apoptotic cells of stem cells. The expression of mRNA in gtf2h2 and prkdc decreased. Conclusion: 1. Gamma-aminobutyric acid signaling system exists in intestinal epithelium. 緯 -aminobutyric acid is involved in regulating the proliferation of intestinal epithelial cells through its type A receptor. Apoptosis and differentiation. Gamma-aminobutyric acid participates in regulating the proliferation of intestinal epithelial cells by its type B receptor. Inhibiting GABA type A receptor or type B receptor can protect the intestinal injury induced by chemotherapeutic drugs.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R574

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