缝隙连接蛋白43参与脑血管痉挛的体外实验研究
发布时间：2018-07-15 21:31
【摘要】：目的： 缝隙连接（gap junction，GJ）是相邻细胞间最直接快速的通讯方式，对维持血管细胞间的协同反应起重要作用。在血管系统中共发现4种缝隙连接蛋白（connexion，Cx），包括Cx37、Cx40、Cx43和Cx45，其中Cx43最为重要，Cx43表达的紊乱，将会导致一系列的病理反应及疾病。前期研究表明Cx43及其相关缝隙连接通道与脑血管痉挛（Cerebral vasospasm，CVS）关系密切，但机制尚不清楚。本课题在前期研究的基础上，对Cx43参与CVS的机制进行深入研究，以期通过体外细胞实验初步探明Cx43参与CVS的相关分子机制。 材料与方法： 1.原代大鼠基底动脉平滑肌细胞（SMCs）的培养和鉴定：分离SD-大鼠基底动脉，利用组织贴块法进行平滑肌细胞原代培养，采用细胞免疫荧光技术对其进行鉴定。 2.shRNA-Cx43腺病毒载体的构建及感染效率的检测：构建质粒，利用293A细胞进行包装和滴度检测；用不同稀释度的病毒对平滑肌细胞进行感染，综合细胞生长状态以及荧光表达量来判断具有最佳作用效果的病毒稀释度，并以此稀释度作为后续试验的依据。同时，采用Western blot检测病毒感染平滑肌细胞后Cx43表达水平的变化。 3.脑血管痉挛体外细胞模型的构建及脑血管痉挛后细胞间收缩信号交流的改变：利用OxyHb刺激平滑肌细胞构建脑血管痉挛双层共培养模型，，在此基础上采用Wester blot技术检测平滑肌细胞Cx43表达的变化，采用HE染色观察平滑肌细胞长度的变化，并用共聚焦显微镜观察并检测Calcein AM、Ca2+、IP3在SMC与SMC、SMC与EC间缝隙连接的交流情况。 4.Cx43表达量的降低对脑血管痉挛体外模型信号通讯的影响：利用shRNA-Cx43腺病毒感染平滑肌细胞，采用同样方法构建脑血管痉挛双层共培养模型，在此基础上采用Wester blot技术检测平滑肌细胞Cx43表达的变化，采用HE染色观察平滑肌细胞长度的变化，并用共聚焦显微镜观察并检测Calcein AM、Ca2+、IP3在SMC与SMC、SMC与EC间缝隙连接的交流情况。 结果： 1.成功培养出原代基底动脉平滑肌细胞，经光镜观察及细胞免疫荧光鉴定，平滑肌细胞形态正确，生长状态良好，纯度较高； 2.成功构建PAV.KdSi.U/eGFP-U6-shCx43质粒，病毒包装7d后可见病毒病变斑（CPE）形成，病毒原液（P0）滴度为1.07×1011PFU/mL，当MOI值取100时，感染效率最高。经Western blot检测发现Cx43在24h表达水平最低，之后基本保持稳定，感染达到预期效果。 3.通过检测正常平滑肌细胞、OxyHb刺激24h、OxyHb刺激48h、OxyHb刺激72h时Cx43表达情况及平滑肌细胞长度，发现OxyHb刺激后Cx43表达量逐渐增加，在48h时到达高峰，之后又逐渐下降。平滑肌细胞长度变化情况与Cx43表达相符，在OxyHb刺激后平滑肌细胞长度缩短，在48h时到达高峰，之后又逐渐增加。当使用腺病毒感染降低平滑肌Cx43的表达后，OxyHb刺激后平滑肌细胞收缩强度有所缓解，长度较未感染前增加。 4.通过激光共聚焦显微镜观察分析双层细胞共培养模型上“受体细胞”的荧光强度，发现shRNA-Cx43腺病毒感染平滑肌后，能够减弱染料Calcein AM和Ca2+在平滑肌细胞间的传输，同时也可以减弱IP3从平滑肌细胞向内皮细胞的传输。各组“对照细胞”均无荧光出现，说明各信号分子由缝隙连接在细胞间传输。 结论： 1.SMCs上Cx43表达随着OxyHb刺激时间的延长逐渐升高，同时SMCs长度逐渐变短，在48h到达高峰，之后逐渐恢复；降低SMCs上Cx43的表达后能有效的缓解平滑肌细胞收缩；Cx43表达量的改变与SMCs收缩有关。 2.Cx43表达量的改变能够影响非特异性物质Calcein AM和收缩因子Ca2+在平滑肌细胞间的交流，同时也会影响IP3在SMCs与VECs间的传输，Cx43表达量越高，缝隙连接对这些物质的通透性越强，反之则弱。 3.总的来说，在脑血管痉挛体外细胞模型中，Cx43的表达量会增高，并且Cx43的表达量与平滑肌细胞收缩强度呈正相关，说明Cx43表达量的改变与平滑肌收缩存在关系，具体机制需后续的深入研究；Cx43表达的增高能够影响平滑肌细胞收缩相关因子在细胞间的交流，Cx43表达越丰富，相关收缩因子的传输性就越强；Cx43参与CVS的机制可能为防治CVS提供新的思路和靶点。
[Abstract]:Objective:
Gap junction (GJ) is the most direct and rapid communication mode between adjacent cells. It plays an important role in maintaining the synergistic response between vascular cells. 4 kinds of connexion (Cx), including Cx37, Cx40, Cx43 and Cx45, are found in the vascular system. Cx43 is the most important, Cx43 expression disorder, which will lead to a series of pathological changes. Reaction and disease. Previous studies have shown that Cx43 and its associated gap junction channel are closely related to Cerebral vasospasm (CVS), but the mechanism is not yet clear. On the basis of previous studies, this topic studies the mechanism of Cx43 participating in CVS in order to preliminarily identify Cx43 to participate in the related molecular machines of CVS through the external cell test. System.
Materials and methods:
1. the culture and identification of the basilar artery smooth muscle cells (SMCs) of the primary rat: isolation of the basilar artery of SD- rats and the primary culture of smooth muscle cells by tissue patch method, and the identification of them by cell immunofluorescence.
Construction of 2.shRNA-Cx43 adenovirus vector and detection of infection efficiency: construct plasmids, use 293A cells for packaging and titer detection, infect smooth muscle cells with different dilution viruses, integrate cell growth state and fluorescence expression to determine the virus dilution with the best use effect, and use this dilution degree as a dilution degree. Western blot was used to detect the expression level of Cx43 after virus infection in smooth muscle cells.
The construction of 3. cerebral vasospasm in vitro cell model and the change of intercellular contractile signal exchange after cerebral vasospasm: using OxyHb to stimulate smooth muscle cells to construct a bilayer co culture model of cerebral vasospasm, based on which Wester blot technique was used to detect the changes of Cx43 expression in smooth muscle cells, and the length of smooth muscle cells was observed by HE staining. The changes of Calcein AM, Ca2+ and IP3 in gap junctions between SMC and SMC, SMC and EC were observed and detected by confocal microscope.
The effect of reduced expression of 4.Cx43 on the signal communication in vitro model of cerebral vasospasm: using shRNA-Cx43 adenovirus to infect smooth muscle cells, the same method was used to construct a double layer co culture model of cerebral vasospasm. On this basis, Wester blot technique was used to detect the changes of Cx43 expression in smooth muscle cells, and HE staining was used to observe the smooth muscle fine. Changes in cell length were observed and confocal microscopy was used to observe and detect Calcein AM, Ca2+ and IP3 exchanges between gap junctions between SMC and SMC, SMC and EC.
Result:
1. the primary basilar artery smooth muscle cells were successfully cultured. Through the light microscopy and the identification of cell immunofluorescence, the smooth muscle cell morphology was correct, the growth state was good and the purity was high.
2. the PAV.KdSi.U/eGFP-U6-shCx43 plasmid was successfully constructed. The virus lesion plaque (CPE) was formed after the virus package 7d. The virus original liquid (P0) titer was 1.07 x 1011PFU/mL. When the MOI value was 100, the infection efficiency was the highest. The Western blot detected the lowest expression level of Cx43 in 24h, and it was basically stable after that, and the infection reached the expected effect.
3. through the detection of normal smooth muscle cells, OxyHb stimulates 24h, OxyHb stimulates 48h, and OxyHb stimulates Cx43 expression and the length of smooth muscle cells. It is found that the Cx43 expression increases gradually after OxyHb stimulation, reaches the peak at 48h, and then gradually decreases. The smooth muscle cell length changes in accordance with Cx43 expression, and the smooth muscle cells after OxyHb stimulate the smooth muscle cells. After the use of adenovirus infection to reduce the expression of smooth muscle Cx43, the contraction intensity of smooth muscle cells was relieved after OxyHb, and the length of OxyHb was increased.
4. the fluorescence intensity of "receptor cells" on the double cell co culture model was observed and analyzed by laser confocal microscopy. It was found that after shRNA-Cx43 adenovirus infected smooth muscle, it could weaken the transmission of dye Calcein AM and Ca2+ in smooth muscle cells, and also reduce the transmission of IP3 from smooth muscle cells to endothelial cells. No fluorescence appeared in the irradiated cells, indicating that the signal molecules were transported through the gap junctions between the cells.
Conclusion:
The expression of Cx43 on 1.SMCs increased gradually with the prolongation of OxyHb stimulation time, and the length of SMCs gradually shortened, then reached the peak at 48h, and then gradually recovered, and the expression of Cx43 in SMCs could effectively alleviate the contraction of smooth muscle cells, and the change of Cx43 expression was related to SMCs contraction.
The changes in the expression of 2.Cx43 can affect the communication between Calcein AM and contractile factor Ca2+ in the non-specific substance, and also affect the transmission of IP3 between SMCs and VECs. The higher the expression of Cx43, the greater the permeability of the gap junction to these substances, and vice versa.
3. in general, the expression of Cx43 increased in the cell model of cerebral vasospasm in vitro, and the expression of Cx43 was positively correlated with the contraction intensity of smooth muscle cells, indicating that the change of Cx43 expression was related to the contraction of smooth muscle, and the specific mechanism needed further study. The increase of Cx43 expression could affect the contractile phase of smooth muscle cells. In the intercellular communication, the more the Cx43 expression is, the greater the transmission of the related contraction factor is, and the mechanism of Cx43 participation in CVS may provide new ideas and targets for the prevention and control of CVS.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743
本文编号：2125440
[Abstract]:Objective:
Gap junction (GJ) is the most direct and rapid communication mode between adjacent cells. It plays an important role in maintaining the synergistic response between vascular cells. 4 kinds of connexion (Cx), including Cx37, Cx40, Cx43 and Cx45, are found in the vascular system. Cx43 is the most important, Cx43 expression disorder, which will lead to a series of pathological changes. Reaction and disease. Previous studies have shown that Cx43 and its associated gap junction channel are closely related to Cerebral vasospasm (CVS), but the mechanism is not yet clear. On the basis of previous studies, this topic studies the mechanism of Cx43 participating in CVS in order to preliminarily identify Cx43 to participate in the related molecular machines of CVS through the external cell test. System.
Materials and methods:
1. the culture and identification of the basilar artery smooth muscle cells (SMCs) of the primary rat: isolation of the basilar artery of SD- rats and the primary culture of smooth muscle cells by tissue patch method, and the identification of them by cell immunofluorescence.
Construction of 2.shRNA-Cx43 adenovirus vector and detection of infection efficiency: construct plasmids, use 293A cells for packaging and titer detection, infect smooth muscle cells with different dilution viruses, integrate cell growth state and fluorescence expression to determine the virus dilution with the best use effect, and use this dilution degree as a dilution degree. Western blot was used to detect the expression level of Cx43 after virus infection in smooth muscle cells.
The construction of 3. cerebral vasospasm in vitro cell model and the change of intercellular contractile signal exchange after cerebral vasospasm: using OxyHb to stimulate smooth muscle cells to construct a bilayer co culture model of cerebral vasospasm, based on which Wester blot technique was used to detect the changes of Cx43 expression in smooth muscle cells, and the length of smooth muscle cells was observed by HE staining. The changes of Calcein AM, Ca2+ and IP3 in gap junctions between SMC and SMC, SMC and EC were observed and detected by confocal microscope.
The effect of reduced expression of 4.Cx43 on the signal communication in vitro model of cerebral vasospasm: using shRNA-Cx43 adenovirus to infect smooth muscle cells, the same method was used to construct a double layer co culture model of cerebral vasospasm. On this basis, Wester blot technique was used to detect the changes of Cx43 expression in smooth muscle cells, and HE staining was used to observe the smooth muscle fine. Changes in cell length were observed and confocal microscopy was used to observe and detect Calcein AM, Ca2+ and IP3 exchanges between gap junctions between SMC and SMC, SMC and EC.
Result:
1. the primary basilar artery smooth muscle cells were successfully cultured. Through the light microscopy and the identification of cell immunofluorescence, the smooth muscle cell morphology was correct, the growth state was good and the purity was high.
2. the PAV.KdSi.U/eGFP-U6-shCx43 plasmid was successfully constructed. The virus lesion plaque (CPE) was formed after the virus package 7d. The virus original liquid (P0) titer was 1.07 x 1011PFU/mL. When the MOI value was 100, the infection efficiency was the highest. The Western blot detected the lowest expression level of Cx43 in 24h, and it was basically stable after that, and the infection reached the expected effect.
3. through the detection of normal smooth muscle cells, OxyHb stimulates 24h, OxyHb stimulates 48h, and OxyHb stimulates Cx43 expression and the length of smooth muscle cells. It is found that the Cx43 expression increases gradually after OxyHb stimulation, reaches the peak at 48h, and then gradually decreases. The smooth muscle cell length changes in accordance with Cx43 expression, and the smooth muscle cells after OxyHb stimulate the smooth muscle cells. After the use of adenovirus infection to reduce the expression of smooth muscle Cx43, the contraction intensity of smooth muscle cells was relieved after OxyHb, and the length of OxyHb was increased.
4. the fluorescence intensity of "receptor cells" on the double cell co culture model was observed and analyzed by laser confocal microscopy. It was found that after shRNA-Cx43 adenovirus infected smooth muscle, it could weaken the transmission of dye Calcein AM and Ca2+ in smooth muscle cells, and also reduce the transmission of IP3 from smooth muscle cells to endothelial cells. No fluorescence appeared in the irradiated cells, indicating that the signal molecules were transported through the gap junctions between the cells.
Conclusion:
The expression of Cx43 on 1.SMCs increased gradually with the prolongation of OxyHb stimulation time, and the length of SMCs gradually shortened, then reached the peak at 48h, and then gradually recovered, and the expression of Cx43 in SMCs could effectively alleviate the contraction of smooth muscle cells, and the change of Cx43 expression was related to SMCs contraction.
The changes in the expression of 2.Cx43 can affect the communication between Calcein AM and contractile factor Ca2+ in the non-specific substance, and also affect the transmission of IP3 between SMCs and VECs. The higher the expression of Cx43, the greater the permeability of the gap junction to these substances, and vice versa.
3. in general, the expression of Cx43 increased in the cell model of cerebral vasospasm in vitro, and the expression of Cx43 was positively correlated with the contraction intensity of smooth muscle cells, indicating that the change of Cx43 expression was related to the contraction of smooth muscle, and the specific mechanism needed further study. The increase of Cx43 expression could affect the contractile phase of smooth muscle cells. In the intercellular communication, the more the Cx43 expression is, the greater the transmission of the related contraction factor is, and the mechanism of Cx43 participation in CVS may provide new ideas and targets for the prevention and control of CVS.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743
【参考文献】
相关期刊论文 前5条
1 陈红伟;洪涛;宋湖平;叶新运;汪阳;;缝隙连接蛋白43磷酸化在兔脑血管痉挛中的表达变化[J];重庆医学;2010年09期
2 叶新运;洪涛;宋湖平;陈红伟;吴远水;;实验性蛛网膜下腔出血后脑血管痉挛兔基底动脉Cx43蛋白表达的时相变化[J];中风与神经疾病杂志;2009年06期
3 洪涛,Hill CE;缝隙连结在蛛网膜、软脑膜及血管浆膜层中的表达[J];中华实验外科杂志;2003年05期
4 洪涛;冯九庚;蒋丽萍;段剑;汪阳;江志群;;RNA干扰抑制血管平滑肌细胞缝隙连接Cx43介导的细胞间通讯[J];中华实验外科杂志;2006年06期
5 洪涛;段剑;汪阳;蒋丽萍;冯九庚;江志群;;一种可应用于研究脑血管痉挛的新型细胞模型[J];中华实验外科杂志;2006年10期
本文编号：2125440
本文链接：https://www.wllwen.com/yixuelunwen/shenjingyixue/2125440.html
最近更新
教材专著
