本文選題：頂細(xì)胞 + 壁細(xì)胞　； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2009年博士論文

【摘要】： 胃粘膜上皮細(xì)胞具有很高的更新速度,各種上皮細(xì)胞具有不同的代謝周期,處于一種動態(tài)的、精致的平衡之中,這種平衡的打破導(dǎo)致各種疾病的發(fā)生。胃粘膜由許多胃單位構(gòu)成的,每個胃單位由頂細(xì)胞(pit cells)、壁細(xì)胞(parietal cells)、主細(xì)胞(chief cells)、頸粘液細(xì)胞(mucous neck cells)、多能干細(xì)胞(multipotent stem cells)以及少量的內(nèi)分泌細(xì)胞(enteroendocrine cells)等多種上皮細(xì)胞組成,其中頂細(xì)胞、壁細(xì)胞、主細(xì)胞是胃粘膜最主要的三種細(xì)胞。各種上皮細(xì)胞均來源于峽部的多能干細(xì)胞,每種細(xì)胞具有不同的功能和更新速率。細(xì)胞分化、增殖和凋亡的改變以及更新速度的改變直接影響到胃粘膜結(jié)構(gòu)的穩(wěn)定,是導(dǎo)致胃相關(guān)疾病發(fā)生的原因。 頂細(xì)胞位于粘膜層的最外側(cè),與胃腔的外界環(huán)境直接接觸,表面覆蓋著從細(xì)胞釋放出來的粘液,有保護(hù)細(xì)胞免受胃液內(nèi)高濃度鹽酸和胃蛋白酶損傷的作用,是胃粘膜的第一道屏障,每3天更新一次。壁細(xì)胞屬于高度特化的終末細(xì)胞,主要功能是分泌鹽酸,位于胃腺腔的頸部和體部,約占胃粘膜上皮細(xì)胞總數(shù)的30%,每隔54天更新一次,是上皮細(xì)胞中體積最大、數(shù)量最多的細(xì)胞。主細(xì)胞又稱胃酶細(xì)胞(zymogenic cells)位于胃腺腔的底部,約190天更換一次,可分泌胃蛋白酶原,并被壁細(xì)胞分泌的鹽酸激活,成為具有活性的胃蛋白酶。這三種細(xì)胞占粘膜層上皮細(xì)胞的絕大部分,是粘膜層的主要細(xì)胞成分。 基于Cre-LoxP系統(tǒng)的組織特異性條件基因敲除技術(shù)是研究基因在特定組織、特定細(xì)胞生理功能的有效手段。目前在胃的各種上皮細(xì)胞中特異性表達(dá)Cre重組酶的轉(zhuǎn)基因小鼠非常少見,在某種程度上妨礙了對調(diào)節(jié)胃正常發(fā)育以及胃相關(guān)疾病遺傳機(jī)制的研究。為了深入研究調(diào)控胃粘膜層上皮細(xì)胞功能的遺傳機(jī)制及其在胃粘膜穩(wěn)態(tài)維持以及胃組織相關(guān)疾病中的功能,我們分別構(gòu)建了在頂細(xì)胞、壁細(xì)胞、主細(xì)胞特異性表達(dá)Cre重組酶的轉(zhuǎn)基因小鼠。 組織細(xì)胞特異性啟動子的選擇是構(gòu)建組織細(xì)胞特異性表達(dá)Cre重組酶轉(zhuǎn)基因小鼠的關(guān)鍵。鈣離子激活蛋白酶(Calpains)是受Ca2+調(diào)節(jié)的半胱氨酸蛋白酶家族,該家族在哺乳動物中有14個成員,其中的7個表達(dá)具有明顯的組織特異性。NCL-2/calpain-8 (Capn8)在胃粘膜層頂細(xì)胞特異性表達(dá)。H+,K+-ATP酶β亞單位(β-subunit of H+, K+-ATPase,Atp4b)是壁細(xì)胞的標(biāo)志物分子。胃蛋白酶原C(Pepsinogen C,Pgc)在胃粘膜的主細(xì)胞特異性表達(dá),是主細(xì)胞的標(biāo)志物分子。因此,本研究擬分別采用Capn8,Atp4b和Pgc基因的啟動子,實(shí)現(xiàn)Cre重組酶在頂細(xì)胞、壁細(xì)胞和主細(xì)胞的特異性表達(dá)。 我們利用PCR擴(kuò)增的方法獲得了3.9-kb Capn8和1.0-kb Atp4b以及2.1-kb Pgc基因的啟動子。將獲得的啟動子連入含有1.2-kb編碼Cre重組酶的基因和2.1-kb人生長激素基因多聚腺苷酸加A信號(hGH)的載體中,得到轉(zhuǎn)基因載體。轉(zhuǎn)基因載體經(jīng)限制性內(nèi)切酶線性化后,經(jīng)顯微注射引入小鼠受精卵。將顯微注射后的受精卵移植入假孕母鼠,從子代小鼠中鑒定得到Capn8-Cre,Atp4b-Cre和Pgc-Cre三種轉(zhuǎn)基因小鼠。 通過將三種胃組織特異性Cre轉(zhuǎn)基因小鼠與Smad4條件基因打靶小鼠(Smad4Co/Co)和ROSA26報告小鼠雜交檢測Cre重組酶表達(dá)的時空分布。提取Capn8-Cre;Smad4Co/+小鼠各組織基因組DNA,用Smad4基因特異引物進(jìn)行PCR,檢測Cre重組酶介導(dǎo)的重組及其組織特異性。結(jié)果顯示,Cre重組酶可在胃介導(dǎo)Smad4基因的敲除并產(chǎn)生特異的陽性條帶。此外,在肝臟和皮膚也有Cre重組酶的表達(dá)。Capn8-Cre;Smad4Co/Co小鼠各組織基因組DNA的Southern Blot雜交鑒定結(jié)果也證實(shí)Cre重組酶在胃、肝臟及皮膚中表達(dá)。通過LacZ染色檢測Capn8-Cre;ROSA26雙轉(zhuǎn)基因小鼠中Cre重組酶表達(dá)的組織及細(xì)胞類型。結(jié)果顯示,Capn8-Cre重組酶在胃粘膜層頂細(xì)胞特異性表達(dá)。除此之外,在肝臟的肝細(xì)胞及皮膚少數(shù)角質(zhì)細(xì)胞也有表達(dá)。Capn8-Cre;Smad4Co/Co基因敲除小鼠胃粘膜層Smad4免疫組織化學(xué)染色結(jié)果也顯示,Capn8-Cre重組酶在頂細(xì)胞介導(dǎo)了Smad4的基因敲除。 利用同樣的方法我們檢測了Atp4b-Cre和Pgc-Cre轉(zhuǎn)基因小鼠Cre重組酶的組織表達(dá)譜。Pgc-Cre轉(zhuǎn)基因小鼠除在胃粘膜主細(xì)胞特異性表達(dá)Cre重組酶外,在十二指腸也有表達(dá)。Atp4b-Cre轉(zhuǎn)基因小鼠僅在胃粘膜壁細(xì)胞特異性表達(dá)Cre重組酶。 為進(jìn)一步驗(yàn)證Cre重組酶的有效性,我們利用Capn8-Cre和Pgc-Cre轉(zhuǎn)基因小鼠與Smad4Co/Co小鼠交配,獲得了胃頂細(xì)胞特異性Smad4基因敲除小鼠( Capn8-Cre;Smad4Co/Co )和胃主細(xì)胞特異性Smad4基因敲除小鼠(Pgc-Cre;Smad4Co/Co),并初步觀察到Smad4基因敲除導(dǎo)致胃粘膜層發(fā)生的組織學(xué)改變。 總之,我們利用3.9-kb Capn8基因啟動子,1.0-kb Atp4b基因啟動子和2.1-kb Pgc基因啟動子成功地構(gòu)建了在胃粘膜層頂細(xì)胞、壁細(xì)胞和主細(xì)胞特異性表達(dá)Cre重組酶的轉(zhuǎn)基因小鼠。研究結(jié)果證明這三種胃組織細(xì)胞特異性Cre重組酶轉(zhuǎn)基因小鼠能夠介導(dǎo)被LoxP序列錨定的基因在相應(yīng)胃粘膜細(xì)胞中被有效剔除。這些轉(zhuǎn)基因小鼠的成功研制為研究胃粘膜上皮細(xì)胞的譜系分化以及相關(guān)細(xì)胞的遺傳控制機(jī)制提供了理想的工具。
[Abstract]:The epithelial cells of the gastric mucosa have a high rate of renewal, and the various epithelial cells have different metabolic cycles and are in a dynamic, delicate balance. This balance breaks the occurrence of various diseases. The gastric mucosa is composed of many gastric units, each of which is pit cells, parietal cells, and main cell. (chief cells), cervical mucous cells (mucous neck cells), pluripotent stem cells (multipotent stem cells) and a small number of endocrine cells (enteroendocrine cells), such as a variety of epithelial cells, of which the top cells, wall cells, main cells are the most important three kinds of cells in the gastric mucosa. The cells have different functions and rates of renewal. The changes in cell differentiation, proliferation and apoptosis and the change of the rate of renewal directly affect the stability of the structure of the gastric mucosa, which is the cause of the occurrence of gastric related diseases.
The parietal cells are located at the most outside of the mucous layer and directly contact with the external environment of the gastric cavity. The surface covers the mucus released from the cells. It protects the cells from the high concentration of hydrochloric acid and pepsin in the gastric juice. It is the first barrier of the gastric mucosa and is updated every 3 days. Parietal cells belong to highly specialized terminal cells. The main work is the main work. The ability to secrete hydrochloric acid, located in the neck and body of the gastric gland cavity, about 30% of the total number of epithelial cells in the gastric mucosa, once every 54 days, is the largest and most large cell in the epithelial cells. The main cell is called zymogenic cells at the bottom of the gastric gland cavity and is replaced about 190 days, which can secrete pepsinogen and be thin wall. The secretion of hydrochloric acid activates and activates pepsin. These three cells account for the vast majority of mucosal epithelial cells and are the major cell components of the mucosa.
Tissue specific conditional gene knockout based on Cre-LoxP system is an effective means to study genes in specific tissues and specific cell physiological functions. At present, transgenic mice that specifically express Cre recombinant enzymes in various epithelial cells of the stomach are very rare. To a certain extent, it hinders the regulation of normal development of the stomach and the related diseases of the stomach. In order to study the genetic mechanism of the function of the epithelial cell of the gastric mucosa and its function in the homeostasis of gastric mucosa, and the function of the gastric tissue related diseases, we constructed the transgenic mice with the expression of Cre recombinant enzyme in the apical cell, the wall cell and the main cell specific expression.
The selection of tissue cell specific promoter is the key to the construction of tissue cell specific expression of Cre recombinant transgenic mice. Calcium activated proteinase (Calpains) is a family of cysteine protease regulated by Ca2+. The family has 14 members in mammals, and 7 of them have obvious tissue specific.NCL-2/calpain-. 8 (Capn8) is a specific expression of.H+ in the parietal cells of the gastric mucosa, and the K+-ATP enzyme beta subunit (beta -subunit of H+, K+-ATPase, Atp4b) is a marker molecule of the wall cells. The pepsinogen C (Pepsinogen C, Pgc) is expressed specifically in the main cells of the gastric mucosa and is a marker molecule of the main cells. The promoter of Cre can express the specific expression of the recombinant enzyme in the parietal cells, parietal cells and main cells.
We obtained the promoter of 3.9-kb Capn8 and 1.0-kb Atp4b and 2.1-kb Pgc gene by PCR amplification. The promoter connected into the vector containing 1.2-kb encoded Cre recombinant enzyme and 2.1-kb human growth hormone gene polyadenyl adenylate plus A signal (hGH). The transgenic vector was restricted by restriction endonuclease. After linearization, the mouse fertilized eggs were injected into the mice by microinjection. The fertilized eggs after microinjection were transplanted into the pregnant mice, and three transgenic mice of Capn8-Cre, Atp4b-Cre and Pgc-Cre were identified from the offspring mice.
The spatio-temporal distribution of the expression of Cre recombinant enzyme was detected by hybridization between three gastric tissue specific Cre transgenic mice and Smad4 conditional gene targeting mice (Smad4Co/Co) and ROSA26. Capn8-Cre, genomic DNA of every tissue of Smad4Co/+ mice and PCR with Smad4 specific primers were used to detect the recombination and tissue specific of Cre recombinant enzyme. The results showed that the Cre recombinant enzyme could be knocked out of the stomach mediated Smad4 gene and produce a specific positive band. In addition, the expression of the Cre recombinant enzyme was.Capn8-Cre in the liver and the skin; the Southern Blot hybridization identification results of the genomic DNA in the Smad4Co/Co mice also confirmed that the Cre heavy enzyme was expressed in the stomach, the liver and the skin. The tissue and cell types of Cre recombinant enzyme in Capn8-Cre and ROSA26 double transgenic mice were detected. The results showed that the Capn8-Cre recombinase was expressed specifically in the gastric mucosal layer apical cells. In addition, the.Capn8-Cre was expressed in the liver cells and the few keratinocytes in the skin, and the Smad4 immune group in the gastric mucosa layer of the Smad4Co/Co gene knockout mice. The results of chemical staining also showed that Capn8-Cre recombinase mediated Smad4 knockout in the apical cells.
Using the same method, we detected the tissue expression profiles of Cre recombinant enzyme of Atp4b-Cre and Pgc-Cre transgenic mice..Pgc-Cre transgenic mice expressed.Atp4b-Cre transgenic mice in the duodenum, except for the specific expression of Cre recombinant enzyme in the main gastric mucosa, and the expression of Cre recombinant enzyme in the gastric mucosa only in the duodenum.
In order to further verify the effectiveness of the Cre recombinant enzyme, we use Capn8-Cre and Pgc-Cre transgenic mice to mate with Smad4Co/Co mice, and obtain the specific Smad4 gene knockout mice (Capn8-Cre; Smad4Co/Co) and the specific Smad4 gene knockout mice (Pgc-Cre; Smad4Co/Co) of the gastric main cell (Pgc-Cre; Smad4Co/Co), and preliminarily observe the Smad4 gene knockout guide. Histologic changes in the mucosa of the gastric mucosa.
In conclusion, we successfully constructed transgenic mice with the 3.9-kb Capn8 gene promoter, the 1.0-kb Atp4b promoter and the 2.1-kb Pgc gene promoter in the gastric mucosa, the wall cells and the main cells specifically expressed the Cre recombinant enzyme. The results showed that the three gastric tissue cell specific Cre recombinase transgenic mice could mediate. The genes that are anchored by the LoxP sequence are effectively eliminated in the corresponding gastric mucosa cells. The successful development of these transgenic mice provides an ideal tool for the study of the genealogical differentiation of gastric epithelial cells and the genetic control mechanism of the related cells.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R346

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 王博婧;胃上皮細(xì)胞Smad4基因敲除導(dǎo)致小鼠胃癌發(fā)生[D];重慶師范大學(xué);2013年

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本文編號：1886201