本文關(guān)鍵詞： 大鼠 再生 晶狀體 干細(xì)胞 信號通路　出處：《青島大學(xué)》2012年博士論文　論文類型：學(xué)位論文

【摘要】：目的： 改進(jìn)大鼠晶狀體再生模型,觀察晶狀體再生過程中干細(xì)胞標(biāo)志物的表達(dá),并初步探討Notch信號通路在晶狀體干細(xì)胞樣細(xì)胞增殖分化中的作用。 方法： 第一部分：大鼠晶狀體再生模型的改進(jìn)對6-8周成年健康Wistar大鼠一眼采用透明角膜切口行晶狀體囊外摘除術(shù),術(shù)中不采用環(huán)形撕囊,而只將晶狀體前囊膜做1/3象限的弧形切開,分離出晶狀體。術(shù)后即刻、1天、3天、1周、2周、1月,分別裂隙燈下觀察晶狀體再生情況并照相,HE染色行病理組織學(xué)檢查。 第二部分：晶狀體再生過程中干細(xì)胞標(biāo)記物的表達(dá)及定位 1.將晶狀體囊外摘除術(shù)后3天、1周、2周及1月的晶狀體再生組織分別取出,提取RNA并反轉(zhuǎn)錄為cDNA,然后通過熒光定量PCR的方法檢測再生組織中干細(xì)胞標(biāo)記物ABCG2、Nestin、PCNA及Telomerase的表達(dá),確定再生晶狀體組織中干細(xì)胞樣細(xì)胞是否存在。設(shè)對側(cè)未手術(shù)眼為正常對照。 2.晶狀體囊外摘除術(shù)后即刻,將晶狀體囊袋組織分為前囊及后囊(包含赤道部)兩部分,分別提取RNA并通過熒光定量PCR的方法檢測各部分干細(xì)胞標(biāo)記物的表達(dá),初步定位晶狀體干細(xì)胞樣細(xì)胞。 3.石蠟包埋的大鼠眼作厚度為4微米的石蠟切片,免疫組化檢測ABCG2及PCNA的表達(dá),對晶狀體干細(xì)胞樣細(xì)胞進(jìn)一步定位。 4.晶狀體囊外摘除術(shù)前3天每天兩次注射BrdU,最后一次注射后行一眼的晶狀體囊外摘除術(shù),術(shù)后即刻、術(shù)后1天、3天及1周分別取眼球石蠟包埋后切片,通過免疫組化的方法檢測BrdU的表達(dá),確定增殖的細(xì)胞所在,輔助定位晶狀體干細(xì)胞樣細(xì)胞。 第三部分：Notch信號通路與晶狀體再生 1.將晶狀體囊外摘除術(shù)后不同時(shí)間點(diǎn)的晶狀體再生組織取出,提取RNA并反轉(zhuǎn)錄為cDNA,然后通過熒光定量PCR的方法檢測Notch信號通路信號分子Notch1、Notch2及Jag1的表達(dá),并且通過對細(xì)胞周期調(diào)控蛋白CyclinDl的PCR檢測,初步驗(yàn)證此信號通路是否存在于晶狀體再生過程中。 2.晶狀體囊外摘除術(shù)后即刻、3天及1周,將再生晶狀體組織取出,分別提取組織蛋白,通過Western blot的方法檢測各部分組織中Notch1、Notch2及Jag1的表達(dá)及變化。 結(jié)果： 第一部分：大鼠晶狀體再生模型的改進(jìn) 大鼠晶狀體囊外摘除術(shù)后,所有手術(shù)眼均很快發(fā)生晶狀體組織再生,其程度隨時(shí)間推移逐漸明顯。至術(shù)后1月,已形成典型的再生晶狀體,其中遠(yuǎn)離囊膜切開處的再生晶狀體透明度高,但囊膜切開處混濁明顯。 晶狀體囊外摘除術(shù)后即刻,僅前囊膜下及赤道部殘留少量單層晶狀體上皮細(xì)胞(LEC)；術(shù)后1天,赤道部殘留晶狀體上皮細(xì)胞開始增生。術(shù)后3天,前后囊間的囊袋內(nèi)布滿增生的晶狀體上皮細(xì)胞,前囊切開處兩斷端翻折伴晶狀體上皮細(xì)胞異常增生。術(shù)后1周,囊袋內(nèi)開始充滿早期增生的晶狀體纖維,但增生的晶狀體上皮細(xì)胞仍緊貼后囊膜。術(shù)后2周,晶狀體前囊及赤道部的上皮細(xì)胞明顯增生,囊袋內(nèi)的晶狀纖維向前伸長呈帶狀,其細(xì)胞核已遠(yuǎn)離后囊膜；在前囊切開的地方,前囊彎曲皺折,細(xì)胞異常增生,前后囊貼合緊密處亦充滿增生細(xì)胞,而兩側(cè)的晶狀體囊袋相對完整,細(xì)胞增生分化排列規(guī)則,內(nèi)側(cè)前后囊結(jié)合處細(xì)胞增生分化類似正常晶體的赤道部,整個(gè)再生的晶狀體類似葫蘆狀。術(shù)后1月,再生的晶狀體纖維填充整個(gè)囊袋,赤道部形成典型的弓形帶,與正常晶狀體赤道部形態(tài)類似。而在前囊切開的部分,晶狀體上皮細(xì)胞增生而無晶狀體纖維形成,兩側(cè)的葫蘆狀再生晶狀體排列規(guī)則,各自形成相對完整的前囊、赤道部以及填充囊袋的晶狀體纖維。第二部分：晶狀體再生過程中干細(xì)胞標(biāo)志物的表達(dá)及定位 熒光定量PCR的方法檢測到正常晶狀體及術(shù)后不同時(shí)間點(diǎn)的再生晶狀體均有組織干細(xì)胞標(biāo)記物信號的存在。晶狀體囊外摘除術(shù)后即刻,將前囊及包含赤道部的后囊分開分別提取RNA后行PCR檢測,兩部分均有干細(xì)胞標(biāo)記物信號的擴(kuò)增。 選取四種干細(xì)胞標(biāo)記物中的兩種PCNA及ABCG2為代表行組織的免疫組化檢測。在晶狀體再生過程中,晶狀體前囊及赤道部均可檢測到兩種信號標(biāo)記的存在,特別是前囊切開的部分,晶狀體上皮細(xì)胞異常增生,而此處PCNA及ABCG2的表達(dá)明顯增多。 腹腔注射BrdU后建模,然后通過免疫組化的方法分別進(jìn)行檢測。結(jié)果顯示,正常成熟晶狀體組織中赤道部及前囊均散在少量BrdU陽性細(xì)胞,注藥后1天即可檢測到,術(shù)后1周仍可見。而在晶狀體再生組織中,術(shù)后即刻赤道部及前囊即可見BrdU陽性細(xì)胞。術(shù)后1天,赤道部晶狀體上皮細(xì)胞開始增生,BrdU陽性細(xì)胞亦開始增多,盡管前囊晶狀體上皮細(xì)胞呈單層,但亦散在少量BrdU陽性細(xì)胞。術(shù)后3天,赤道部及前囊均即可見較多BrdU陽性細(xì)胞,特別是前囊切開的地方,晶狀體上皮細(xì)胞異常增生,相應(yīng)BrdU陽性細(xì)胞亦較多。術(shù)后1周時(shí),赤道部及前囊仍可見BrdU陽性細(xì)胞,但較術(shù)后3天明顯減少。第三部分：Notch信號通路在晶狀體再生中的表達(dá)及作用 正常晶狀體組織及再生晶狀體組織中均檢測到Notch通路受體Notch1、Notch2及Notch配體Jag1的表達(dá),且晶狀體囊外摘除術(shù)后早期表達(dá)量呈上升趨勢,術(shù)后1周表達(dá)量最高,之后逐漸降低。而相應(yīng)的細(xì)胞周期調(diào)控關(guān)鍵蛋白CyclinD1的表達(dá)趨勢與此類似。 根據(jù)PCR結(jié)果,選取術(shù)后1周內(nèi)的晶狀體再生組織行Western blot檢測,結(jié)果顯示術(shù)后即刻即有Notch2的表達(dá),術(shù)后3天Notch2表達(dá)明顯,術(shù)后1周表達(dá)降低,而Notch1術(shù)后1周表達(dá)明顯。Jag1在術(shù)后3天開始表達(dá),術(shù)后1周表達(dá)量明顯。但作為對照的正常晶狀體組織未檢測到各信號分子蛋白的表達(dá)。 結(jié)論： 1改進(jìn)的大鼠晶狀體再生模型短期內(nèi)即可再生出新的晶狀體組織,且遠(yuǎn)離前囊切開的部分再生組織較透明,相應(yīng)地晶狀體上皮細(xì)胞排列較規(guī)則。而前囊切開部分的晶狀體組織明顯混濁,相應(yīng)晶狀體上皮細(xì)胞異常增生,未發(fā)生晶狀體纖維化改變。 2正常晶狀體及再生晶狀體組織中均存在干細(xì)胞樣細(xì)胞,其在晶狀體再生過程中發(fā)揮重要作用。正常的晶狀體組織內(nèi)僅少量的干細(xì)胞樣細(xì)胞存在,以維持晶狀體的自我更新；但在晶狀體損傷后的再生過程中,前囊以及赤道部均有干細(xì)胞樣細(xì)胞發(fā)揮作用,從而迅速發(fā)生增殖分化,再生出新的晶狀體組織。 3 Notch信號通路于晶狀體再生的早期即被激活,調(diào)控晶狀體干細(xì)胞樣細(xì)胞的增殖。
[Abstract]:Purpose : To improve the rat lens regeneration model and to observe the expression of stem cell markers in lens regeneration , and to investigate the role of Notch signaling pathway in the proliferation and differentiation of lens stem cell - like cells . Method : In the first part , the rat lens regeneration model was improved for 6 - 8 weeks of adult healthy Wistar rats . Part 2 : Expression and localization of stem cell marker during lens regeneration 1 . The lens regenerating tissues were taken out , RNA was extracted and reverse transcribed into cDNA for 3 days , 1 week , 2 weeks and 1 month after the lens capsule extirpation , then the expression of ABCG2 , Nestin , PCNA and PCNA was detected by fluorescence quantitative PCR . 2 . The lens capsule was divided into two parts : the anterior capsule and the posterior capsule ( including the equatorial portion ) . RNA was extracted and the expression of the marker of each stem cell was detected by fluorescence quantitative PCR . The lens stem cell - like cells were initially located . 3 . Paraffin - embedded rat eyes were sectioned with paraffin sections with a thickness of 4 microns . The expression of ABCG2 and PCNA was detected by immunohistochemistry . 4 . After the lens capsule extirpation was performed twice daily for 3 days , the posterior lens capsule was injected twice a day , and then the posterior lens capsule was taken out after the last injection . The paraffin - embedded sections of the eyeball were taken respectively at 1 day , 3 days and 1 week after the operation , and the expression of the cells was detected by immunohistochemistry . The proliferation of the cells was determined , and the lens stem cells like cells were located in the auxiliary position . Part III : Notch signaling pathway and lens regeneration 1 . The lens regenerative tissues at different time points were taken out , RNA was extracted and reverse transcribed into cDNA , then the expression of Notch1 , Notch2 and Jag1 was detected by fluorescence quantitative PCR . 2 . Immediate , 3 - day and 1 - week post - cataract extraction , the tissue protein was extracted and the expression and change of Notch1 , Notch2 and Jag1 in each tissue were detected by Western blot . Results : Part I : Improvement of Rat Lens Regeneration Model The lens tissue regeneration was observed in all the eyes of the lens after cataract extraction in rats . The degree of lens tissue regeneration was gradually increased over time . By January , the lens was formed with a typical regeneration lens , in which the lens had a higher transparency than the regeneration lens at the incision of the capsule , but the opacity of the capsule was obvious . The lens epithelial cells of the anterior capsule and the equatorial portion of the lens were proliferated . The lens epithelial cells in the anterior capsule and the equatorial region were proliferated . The lens fibers of the anterior capsule and the anterior capsule were more complete . The lens fibers in the anterior and posterior capsule were more complete . The lens fibers in the anterior and posterior capsule were more complete . The fluorescence quantitative PCR method was used to detect the presence of marker signal of tissue stem cells in both the normal lens and the regenerated lens at different time points after operation . The anterior capsule and the posterior capsule containing the equatorial portion were separated from the posterior capsule containing the equatorial portion , and then the stem cell marker signal was amplified . Both PCNA and ABCG2 in four stem cell markers were selected to represent the immunohistochemical detection of the tissue . In the process of lens regeneration , both the anterior capsule and the equatorial portion of the lens could detect the existence of two signal markers , especially those of anterior capsule , and abnormal proliferation of lens epithelial cells , and the expression of PCNA and ABCG2 was significantly increased here . The results showed that both the equatorial region and the anterior capsule of the normal mature lens were scattered in a small number of cells . After 1 day of operation , the cells of the equatorial region and the anterior capsule could be seen . The expression of Notch1 , Notch2 and Notch ligand Jag1 was detected in both the normal lens tissue and the regeneration lens tissue , and the early expression level of the lens capsule was increased . The expression level was highest at 1 week postoperatively , and the expression of Cyclin D1 was similar to that of the corresponding cell cycle regulation key protein Cyclin D1 . According to the results of PCR , Western blot was used to detect the expression of Notch2 in 1 week after surgery . The expression of Notch2 was significantly lower in 3 days after operation . The expression of Notch2 was significantly decreased at 1 week after operation . Conclusion : 1 . The improved lens regeneration model can regenerate the new lens tissue in the short term , and the partial regeneration tissues away from the anterior capsule are more transparent , and the lens epithelial cells are arranged more regularly . There are stem cell - like cells in both normal and regenerative lens tissues . It plays an important role in lens regeneration . Only a small amount of stem cell - like cells exist in the normal lens tissues to maintain the self - renewal of the lens . However , in the regeneration process after lens injury , the stem cells - like cells play a role in the anterior capsule and the equatorial portion , thereby rapidly proliferating and differentiating and regenerating the new lens tissue . The 3 Notch signaling pathway is activated at the early stage of lens regeneration and regulates the proliferation of lens stem cell - like cells .

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R779.6;R-332

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