siRNA干擾Cx37對鼠胃癌皮下移植瘤的影響
發(fā)布時間：2018-12-15 16:15
【摘要】：第一部分篩選Cx37基因的有效si RNA干擾序列目的:篩選有效Cx37-si RNA干擾序列。方法:根據(jù)Cx37基因序列設(shè)計si RNA干擾片段,與Cx37基因過表達(dá)載體共轉(zhuǎn)染HEK293細(xì)胞,檢測Cx37基因蛋白表達(dá)水平,確定有效Cx37-si RNA干擾序列。結(jié)果:通過Western-blot分析,從三種不同的Cx37-si RNA中確定了2條可有效抑制Cx37基因蛋白表達(dá)的si RNA序列。選擇Cx37-si RNA-3片段,進(jìn)行下游si RNA干擾序列慢病毒表達(dá)載體及慢病毒顆粒的構(gòu)建。結(jié)論:成功篩選Cx37基因的有效si RNA干擾序列。第二部分構(gòu)建攜帶Cx37-si RNA有效干擾序列載體的慢病毒顆粒及MFC胃癌細(xì)胞小鼠皮下移植瘤模型目的:構(gòu)建MFC胃癌細(xì)胞小鼠皮下移植瘤模型,向瘤體注射攜帶Cx37-si RNA有效干擾序列載體的慢病毒顆粒,并確定Cx37基因表達(dá)的抑制情況。方法:利用RPMI 1640完全培養(yǎng)基培養(yǎng)MFC小鼠胃癌細(xì)胞系。在30只雌性BALB/c-nu/nu小鼠前肢皮下接種0.2 ml MFC小鼠胃癌細(xì)胞懸液(2.5×107cells/ml),建立皮下移植瘤模型。將小鼠隨機(jī)分成3組,分別向?qū)嶒炇罅鲶w內(nèi)注入攜帶Cx37-si RNA的慢病毒顆粒、攜帶陰性對照-si RNA的慢病毒顆粒和生理鹽水。實(shí)驗終結(jié)時,測量3組小鼠體重及移植瘤。用10%中性福爾馬林溶液固定移植瘤,石蠟包埋后切片,進(jìn)行蘇木精-伊紅染色,并用熒光顯微鏡觀察Cx37-si RNA載體中GFP基因蛋白表達(dá)及分布情況。提取小鼠移植瘤總RNA和總蛋白,通過RT-PCR和Western-blot法檢測Cx37 m RNA和蛋白表達(dá)水平。結(jié)果:利用熒光顯微鏡可見瘤體內(nèi)存在GFP蛋白的大量表達(dá),提示攜帶Cx37-si RNA的慢病毒轉(zhuǎn)染成功。各組小鼠體重?zé)o明顯差異。Cx37-si RNA組移植瘤中Cx37基因m RNA的表達(dá)水平明顯低于mock-si RNA組和生理鹽水組(P0.05),Cx37基因m RNA表達(dá)水平在mock-si RNA組和生理鹽水組之間無明顯差異。Western-blot分析顯示,Cx37-RNAi組移植瘤中Cx37蛋白表達(dá)水平明顯低于mock-si RNA組和生理鹽水組(P0.05)。結(jié)論:向MFC胃癌細(xì)胞小鼠皮下移植瘤中注射攜帶Cx37-si RNA有效干擾序列載體的慢病毒顆粒有效抑制了Cx37基因表達(dá)。第三部分干擾MFC胃癌細(xì)胞小鼠皮下移植瘤中Cx37基因表達(dá)對瘤體細(xì)胞增殖和凋亡的影響目的:明確注射Cx37-si RNA慢病毒對MFC胃癌細(xì)胞小鼠皮下移植瘤瘤體細(xì)胞增殖和凋亡的影響。方法:按照第二部分的實(shí)驗方法,利用40只雌性BALB/c-nu/nu小鼠構(gòu)建MFC胃癌細(xì)胞小鼠皮下移植瘤模型,隨機(jī)分成4組:未注射組、Cx37-si RNA組、mocksi RNA組和生理鹽水組。分別向后三組實(shí)驗鼠瘤體內(nèi)注入攜帶Cx37-si RNA的慢病毒顆粒、攜帶陰性對照-si RNA的慢病毒顆粒和生理鹽水。8周后,利用CCK8方法檢測各組移植瘤的細(xì)胞增殖能力,利用Annexin V-FITC和PI染色方法、Hoechst33342染色方法,以及Bcl-2和Bax m RNA及蛋白表達(dá)水平的檢測,明確瘤體內(nèi)細(xì)胞凋亡情況。結(jié)果:CCK8方法檢測各組移植瘤的細(xì)胞增殖能力結(jié)果顯示,Cx37-si RNA組移植瘤細(xì)胞增殖能力明顯低于其他三個實(shí)驗組(P0.01)。Annexin V-FITC和PI染色方法檢測各組移植瘤細(xì)胞凋亡情況結(jié)果發(fā)現(xiàn),Cx37-si RNA組移植瘤中凋亡細(xì)胞數(shù)明顯高于mock-si RNA組和生理鹽水組(P0.05)。Hoechst33342染色實(shí)驗結(jié)果發(fā)現(xiàn),Cx37-si RNA組移植瘤中細(xì)胞核濃縮的陽性細(xì)胞數(shù)量明顯高于mock-si RNA組和生理鹽水組(P0.01)。RT-PCR及Western-blot實(shí)驗結(jié)果顯示,Cx37-si RNA組移植瘤中Bcl-2和Bax的m RNA和蛋白表達(dá)比值均明顯低于mock-si RNA組和生理鹽水組(P0.01),Mock-si RNA組與生理鹽水組相比Bcl-2/Bax沒有明顯差異。結(jié)論:干擾Cx37基因表達(dá)水平可有效抑制MFC胃癌細(xì)胞小鼠皮下移植瘤細(xì)胞增殖能力,并可促進(jìn)移植瘤細(xì)胞凋亡。
[Abstract]:The first part screens the effective si RNA interference sequence of the Cx37 gene: screening an effective Cx37-si RNA interference sequence. Methods: The expression level of Cx37 gene was detected and the effective Cx37-si RNA interference sequence was determined by co-transfecting the HEK293 cells with the Cx37 gene overexpression vector according to the Cx37 gene sequence. Results: The si-RNA sequence of the expression of Cx37 gene can be effectively inhibited from three different Cx37-si RNA by Western-blot analysis. The Cx37-si RNA-3 fragment was selected for the construction of the downstream si RNA interference sequence lentiviral expression vector and lentiviral particles. Conclusion: The effective si RNA interference sequence of Cx37 gene was successfully screened. the second part constructs the slow virus particles carrying the Cx37-si RNA effective interference sequence carrier and the model of the subcutaneous transplantation of the MFC gastric cancer cell mouse: constructing a subcutaneous transplantation tumor model of the MFC gastric cancer cell mouse, and injecting the slow virus particles carrying the Cx37-si RNA into the tumor body to effectively interfere with the sequence carrier, and the expression of Cx37 gene was determined. Methods: The mouse gastric cancer cell line was cultured with RPMI 1640 medium. In 30 female BALB/ c-nu/ nu mice, 0.2 ml of MFC mouse gastric cancer cell suspension (2.5-107cells/ ml) was subcutaneously inoculated with 0.2 ml of MFC mouse gastric cancer cell suspension (2.5-107cells/ ml). The mice were randomly divided into three groups, and the lentiviral particles carrying Cx37-si RNA were injected into the mouse tumor and the lentiviral particles and physiological saline of negative control-si RNA were carried. At the end of the experiment, the body weight and the transplanted tumor of group 3 mice were measured. The expression and distribution of GFP gene in Cx37-si RNA vector were observed by means of a 10% neutral buffered formalin, paraffin-embedded section, and hematoxylin-eosin staining. The total RNA and total protein of the mice were extracted, and the expression level of Cx37 mRNA and protein was detected by RT-PCR and Western-blot. Results: The expression of GFP in the tumor was detected by fluorescence microscope, and the transfection of the lentivirus with Cx37-si RNA was successful. There was no significant difference in body weight of each group. The expression of Cx37 mRNA in the Cx37-si RNA group was significantly lower than that of the Mock-si RNA group and the normal saline group (P0.05). The expression level of Cx37 gene was not significantly different between the Mock-si RNA group and the normal saline group. Western-blot analysis showed that the expression of Cx37 in Cx37-RNAi group was significantly lower than that of the Mock-si RNA group and the normal saline group (P0.05). Conclusion: The expression of Cx37 gene was effectively inhibited by the injection of the lentiviral particles with the effective interfering sequence vector of Cx37-si RNA into the subcutaneous transplanted tumor of the MFC. The third part of the study on the effect of Cx37 gene expression on the proliferation and apoptosis of the tumor cells in the mice with MFC gastric cancer cells: the effect of the injection of Cx37-si RNA lentivirus on the proliferation and apoptosis of the tumor cells of the mice with MFC gastric cancer cells was well-defined. Methods: According to the experimental method of the second part, 40 female BALB/ c-nu/ nu mice were used to construct the subcutaneous transplantation tumor model of MFC gastric cancer cells, and the mice were randomly divided into 4 groups: the non-injection group, the Cx37-si RNA group, the mcksi RNA group and the normal saline group. The lentiviral particles carrying Cx37-si RNA and the lentiviral particles and physiological saline with negative control-si RNA were injected into the mice of the three experimental mice respectively. After 8 weeks, the cell proliferation ability of each group of transplanted tumors was detected by the CCK8 method, and the Hoechst33342 staining method was used by the method of Annexin V-FITC and PI staining. and the expression level of the Bcl-2 and Bax mRNA and the protein expression level are detected, and the cell apoptosis in the tumor is determined. Results: The results showed that the proliferation ability of Cx37-si RNA group was significantly lower than that of the other three experimental groups (P0.01). The number of apoptotic cells in the Cx37-si RNA group was significantly higher than that of the Mock-si RNA group and the normal saline group (P0.05). The results of the Hoechst33342 staining showed that the number of positive cells in the nucleus of the Cx37-si RNA group was significantly higher than that of the Mock-si RNA group and the normal saline group (P0.01). The results of RT-PCR and Western-blot analysis showed that the number of cells in the Cx37-si RNA group was significantly higher than that of the Mock-si RNA group and the normal saline group (P0.01). The expression of Bcl-2 and Bax in the Cx37-si RNA group was significantly lower than that of the Mock-si RNA group and the normal saline group (P0.01). The Bcl-2/ Bax in the Mock-si RNA group was not significantly different from that of the normal saline group. Conclusion: The interference of Cx37 gene expression level can effectively inhibit the proliferation of the transplanted tumor cells in the mice with MFC, and can promote the apoptosis of the transplanted tumor cells.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735.2
本文編號：2380945
[Abstract]:The first part screens the effective si RNA interference sequence of the Cx37 gene: screening an effective Cx37-si RNA interference sequence. Methods: The expression level of Cx37 gene was detected and the effective Cx37-si RNA interference sequence was determined by co-transfecting the HEK293 cells with the Cx37 gene overexpression vector according to the Cx37 gene sequence. Results: The si-RNA sequence of the expression of Cx37 gene can be effectively inhibited from three different Cx37-si RNA by Western-blot analysis. The Cx37-si RNA-3 fragment was selected for the construction of the downstream si RNA interference sequence lentiviral expression vector and lentiviral particles. Conclusion: The effective si RNA interference sequence of Cx37 gene was successfully screened. the second part constructs the slow virus particles carrying the Cx37-si RNA effective interference sequence carrier and the model of the subcutaneous transplantation of the MFC gastric cancer cell mouse: constructing a subcutaneous transplantation tumor model of the MFC gastric cancer cell mouse, and injecting the slow virus particles carrying the Cx37-si RNA into the tumor body to effectively interfere with the sequence carrier, and the expression of Cx37 gene was determined. Methods: The mouse gastric cancer cell line was cultured with RPMI 1640 medium. In 30 female BALB/ c-nu/ nu mice, 0.2 ml of MFC mouse gastric cancer cell suspension (2.5-107cells/ ml) was subcutaneously inoculated with 0.2 ml of MFC mouse gastric cancer cell suspension (2.5-107cells/ ml). The mice were randomly divided into three groups, and the lentiviral particles carrying Cx37-si RNA were injected into the mouse tumor and the lentiviral particles and physiological saline of negative control-si RNA were carried. At the end of the experiment, the body weight and the transplanted tumor of group 3 mice were measured. The expression and distribution of GFP gene in Cx37-si RNA vector were observed by means of a 10% neutral buffered formalin, paraffin-embedded section, and hematoxylin-eosin staining. The total RNA and total protein of the mice were extracted, and the expression level of Cx37 mRNA and protein was detected by RT-PCR and Western-blot. Results: The expression of GFP in the tumor was detected by fluorescence microscope, and the transfection of the lentivirus with Cx37-si RNA was successful. There was no significant difference in body weight of each group. The expression of Cx37 mRNA in the Cx37-si RNA group was significantly lower than that of the Mock-si RNA group and the normal saline group (P0.05). The expression level of Cx37 gene was not significantly different between the Mock-si RNA group and the normal saline group. Western-blot analysis showed that the expression of Cx37 in Cx37-RNAi group was significantly lower than that of the Mock-si RNA group and the normal saline group (P0.05). Conclusion: The expression of Cx37 gene was effectively inhibited by the injection of the lentiviral particles with the effective interfering sequence vector of Cx37-si RNA into the subcutaneous transplanted tumor of the MFC. The third part of the study on the effect of Cx37 gene expression on the proliferation and apoptosis of the tumor cells in the mice with MFC gastric cancer cells: the effect of the injection of Cx37-si RNA lentivirus on the proliferation and apoptosis of the tumor cells of the mice with MFC gastric cancer cells was well-defined. Methods: According to the experimental method of the second part, 40 female BALB/ c-nu/ nu mice were used to construct the subcutaneous transplantation tumor model of MFC gastric cancer cells, and the mice were randomly divided into 4 groups: the non-injection group, the Cx37-si RNA group, the mcksi RNA group and the normal saline group. The lentiviral particles carrying Cx37-si RNA and the lentiviral particles and physiological saline with negative control-si RNA were injected into the mice of the three experimental mice respectively. After 8 weeks, the cell proliferation ability of each group of transplanted tumors was detected by the CCK8 method, and the Hoechst33342 staining method was used by the method of Annexin V-FITC and PI staining. and the expression level of the Bcl-2 and Bax mRNA and the protein expression level are detected, and the cell apoptosis in the tumor is determined. Results: The results showed that the proliferation ability of Cx37-si RNA group was significantly lower than that of the other three experimental groups (P0.01). The number of apoptotic cells in the Cx37-si RNA group was significantly higher than that of the Mock-si RNA group and the normal saline group (P0.05). The results of the Hoechst33342 staining showed that the number of positive cells in the nucleus of the Cx37-si RNA group was significantly higher than that of the Mock-si RNA group and the normal saline group (P0.01). The results of RT-PCR and Western-blot analysis showed that the number of cells in the Cx37-si RNA group was significantly higher than that of the Mock-si RNA group and the normal saline group (P0.01). The expression of Bcl-2 and Bax in the Cx37-si RNA group was significantly lower than that of the Mock-si RNA group and the normal saline group (P0.01). The Bcl-2/ Bax in the Mock-si RNA group was not significantly different from that of the normal saline group. Conclusion: The interference of Cx37 gene expression level can effectively inhibit the proliferation of the transplanted tumor cells in the mice with MFC, and can promote the apoptosis of the transplanted tumor cells.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735.2
【相似文獻(xiàn)】
相關(guān)期刊論文 前10條
1 李志偉,王占民,吳曉鵬,劉軍,馬道新,劉春生;胞嘧啶脫氨酶基因、單純皰疹病毒胸苷激酶基因治療裸鼠肝門部膽管癌皮下移植瘤的研究[J];中華實(shí)驗外科雜志;2004年10期
2 孫培龍,汪永錄,林萬隆,郭偉劍,胡靜波,劉須才,馬曉明,陳漢;奧曲肽對小白鼠肝癌皮下移植瘤的抑制作用及機(jī)制研究[J];中華肝膽外科雜志;2002年05期
3 王紅衛(wèi);卓忠雄;趙樹文;趙延新;皋月娟;李京京;;大鼠Walker-256皮下移植瘤模型的建立及其超聲評價[J];臨床超聲醫(yī)學(xué)雜志;2007年02期
4 曹雯;林晨輝;;健脾為基礎(chǔ)中藥方對人胃癌MKN-45裸小鼠皮下移植瘤生長及瘤組織內(nèi)P-Stat3蛋白表達(dá)的影響[J];中外醫(yī)療;2013年18期
5 ;基礎(chǔ)理論[J];中國癌癥防治雜志;1985年05期
6 田廣;劉晶晶;房學(xué)東;宮路路;;CD147-shRNA重組質(zhì)粒對裸鼠肝癌細(xì)胞SMMC-7721皮下移植瘤的抑制作用[J];中國實(shí)驗診斷學(xué);2013年10期
7 許建華;鄧皖利;孫玨;張勇;李朝衡;范忠澤;;腸胃清對人大腸癌細(xì)胞HCT8/V皮下移植瘤長春新堿耐藥的逆轉(zhuǎn)作用[J];上海中醫(yī)藥大學(xué)學(xué)報;2009年02期
8 楊一濤;唐羅生;曾祥云;董曉光;李學(xué)喜;袁思奇;劉艷芳;;褪黑素對裸鼠HXO-RB_(44)細(xì)胞皮下移植瘤的抑制作用[J];眼科新進(jìn)展;2009年11期
9 張永紅;唐羅生;;雙自殺基因CDTK載體系統(tǒng)對裸小鼠Y79細(xì)胞皮下移植瘤的殺傷作用[J];國際眼科雜志;2010年10期
10 金泉克;許建華;肖海娟;陸海;余文燕;王國娟;孫玨;范忠澤;;腸胃清煎劑對裸鼠大腸癌皮下移植瘤奧沙利鉑治療的增效作用[J];環(huán)球中醫(yī)藥;2012年03期
相關(guān)博士學(xué)位論文 前1條
1 景元明;siRNA干擾Cx37對鼠胃癌皮下移植瘤的影響[D];蘇州大學(xué);2015年
相關(guān)碩士學(xué)位論文 前8條
1 陳小蕓;腺病毒介導(dǎo)的IL-28B對U14宮頸癌皮下移植瘤小鼠調(diào)節(jié)性T細(xì)胞的影響及抗腫瘤作用的研究[D];蘭州大學(xué);2015年
2 高勝利;白介素18治療裸鼠肝癌皮下移植瘤的初步研究[D];蘇州大學(xué);2008年
3 夏如民;納秒級陡脈沖對卵巢癌細(xì)胞凋亡的外源性通路影響和治療皮下移植瘤安全性研究[D];重慶醫(yī)科大學(xué);2012年
4 鄒金;電穿孔介導(dǎo)Kallistatin基因在體內(nèi)的表達(dá)對NCI-H446皮下移植瘤的預(yù)防和治療效果[D];華僑大學(xué);2012年
5 劉昱良;三七皂甙制劑抑制小鼠胰腺癌皮下移植瘤的實(shí)驗研究[D];昆明醫(yī)科大學(xué);2013年
6 向安東;三七皂甙制劑抑制小鼠膽管癌皮下移植瘤的實(shí)驗研究[D];昆明醫(yī)科大學(xué);2014年
7 韓志俊;Balb/c小鼠皮膚腺癌皮下移植瘤模型的建立及JS-K體內(nèi)抗腫瘤活性的研究[D];遵義醫(yī)學(xué)院;2011年
8 金愛花;漏蘆對小鼠H_（22）肝癌皮下移植瘤的抑制作用及機(jī)制研究[D];延邊大學(xué);2011年
，本文編號：2380945
本文鏈接：http://sikaile.net/yixuelunwen/zlx/2380945.html
最近更新
教材專著
