縫隙連接蛋白43參與腦血管痙攣的體外實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-07-15 21:31
【摘要】：目的： 縫隙連接（gap junction，GJ）是相鄰細(xì)胞間最直接快速的通訊方式，對維持血管細(xì)胞間的協(xié)同反應(yīng)起重要作用。在血管系統(tǒng)中共發(fā)現(xiàn)4種縫隙連接蛋白（connexion，Cx），包括Cx37、Cx40、Cx43和Cx45，其中Cx43最為重要，Cx43表達(dá)的紊亂，將會導(dǎo)致一系列的病理反應(yīng)及疾病。前期研究表明Cx43及其相關(guān)縫隙連接通道與腦血管痙攣（Cerebral vasospasm，CVS）關(guān)系密切，但機(jī)制尚不清楚。本課題在前期研究的基礎(chǔ)上，對Cx43參與CVS的機(jī)制進(jìn)行深入研究，以期通過體外細(xì)胞實(shí)驗(yàn)初步探明Cx43參與CVS的相關(guān)分子機(jī)制。 材料與方法： 1.原代大鼠基底動(dòng)脈平滑肌細(xì)胞（SMCs）的培養(yǎng)和鑒定：分離SD-大鼠基底動(dòng)脈，利用組織貼塊法進(jìn)行平滑肌細(xì)胞原代培養(yǎng)，采用細(xì)胞免疫熒光技術(shù)對其進(jìn)行鑒定。 2.shRNA-Cx43腺病毒載體的構(gòu)建及感染效率的檢測：構(gòu)建質(zhì)粒，利用293A細(xì)胞進(jìn)行包裝和滴度檢測；用不同稀釋度的病毒對平滑肌細(xì)胞進(jìn)行感染，綜合細(xì)胞生長狀態(tài)以及熒光表達(dá)量來判斷具有最佳作用效果的病毒稀釋度，并以此稀釋度作為后續(xù)試驗(yàn)的依據(jù)。同時(shí)，采用Western blot檢測病毒感染平滑肌細(xì)胞后Cx43表達(dá)水平的變化。 3.腦血管痙攣體外細(xì)胞模型的構(gòu)建及腦血管痙攣后細(xì)胞間收縮信號交流的改變：利用OxyHb刺激平滑肌細(xì)胞構(gòu)建腦血管痙攣雙層共培養(yǎng)模型，，在此基礎(chǔ)上采用Wester blot技術(shù)檢測平滑肌細(xì)胞Cx43表達(dá)的變化，采用HE染色觀察平滑肌細(xì)胞長度的變化，并用共聚焦顯微鏡觀察并檢測Calcein AM、Ca2+、IP3在SMC與SMC、SMC與EC間縫隙連接的交流情況。 4.Cx43表達(dá)量的降低對腦血管痙攣體外模型信號通訊的影響：利用shRNA-Cx43腺病毒感染平滑肌細(xì)胞，采用同樣方法構(gòu)建腦血管痙攣雙層共培養(yǎng)模型，在此基礎(chǔ)上采用Wester blot技術(shù)檢測平滑肌細(xì)胞Cx43表達(dá)的變化，采用HE染色觀察平滑肌細(xì)胞長度的變化，并用共聚焦顯微鏡觀察并檢測Calcein AM、Ca2+、IP3在SMC與SMC、SMC與EC間縫隙連接的交流情況。 結(jié)果： 1.成功培養(yǎng)出原代基底動(dòng)脈平滑肌細(xì)胞，經(jīng)光鏡觀察及細(xì)胞免疫熒光鑒定，平滑肌細(xì)胞形態(tài)正確，生長狀態(tài)良好，純度較高； 2.成功構(gòu)建PAV.KdSi.U/eGFP-U6-shCx43質(zhì)粒，病毒包裝7d后可見病毒病變斑（CPE）形成，病毒原液（P0）滴度為1.07×1011PFU/mL，當(dāng)MOI值取100時(shí)，感染效率最高。經(jīng)Western blot檢測發(fā)現(xiàn)Cx43在24h表達(dá)水平最低，之后基本保持穩(wěn)定，感染達(dá)到預(yù)期效果。 3.通過檢測正常平滑肌細(xì)胞、OxyHb刺激24h、OxyHb刺激48h、OxyHb刺激72h時(shí)Cx43表達(dá)情況及平滑肌細(xì)胞長度，發(fā)現(xiàn)OxyHb刺激后Cx43表達(dá)量逐漸增加，在48h時(shí)到達(dá)高峰，之后又逐漸下降。平滑肌細(xì)胞長度變化情況與Cx43表達(dá)相符，在OxyHb刺激后平滑肌細(xì)胞長度縮短，在48h時(shí)到達(dá)高峰，之后又逐漸增加。當(dāng)使用腺病毒感染降低平滑肌Cx43的表達(dá)后，OxyHb刺激后平滑肌細(xì)胞收縮強(qiáng)度有所緩解，長度較未感染前增加。 4.通過激光共聚焦顯微鏡觀察分析雙層細(xì)胞共培養(yǎng)模型上“受體細(xì)胞”的熒光強(qiáng)度，發(fā)現(xiàn)shRNA-Cx43腺病毒感染平滑肌后，能夠減弱染料Calcein AM和Ca2+在平滑肌細(xì)胞間的傳輸，同時(shí)也可以減弱IP3從平滑肌細(xì)胞向內(nèi)皮細(xì)胞的傳輸。各組“對照細(xì)胞”均無熒光出現(xiàn)，說明各信號分子由縫隙連接在細(xì)胞間傳輸。 結(jié)論： 1.SMCs上Cx43表達(dá)隨著OxyHb刺激時(shí)間的延長逐漸升高，同時(shí)SMCs長度逐漸變短，在48h到達(dá)高峰，之后逐漸恢復(fù)；降低SMCs上Cx43的表達(dá)后能有效的緩解平滑肌細(xì)胞收縮；Cx43表達(dá)量的改變與SMCs收縮有關(guān)。 2.Cx43表達(dá)量的改變能夠影響非特異性物質(zhì)Calcein AM和收縮因子Ca2+在平滑肌細(xì)胞間的交流，同時(shí)也會影響IP3在SMCs與VECs間的傳輸，Cx43表達(dá)量越高，縫隙連接對這些物質(zhì)的通透性越強(qiáng)，反之則弱。 3.總的來說，在腦血管痙攣體外細(xì)胞模型中，Cx43的表達(dá)量會增高，并且Cx43的表達(dá)量與平滑肌細(xì)胞收縮強(qiáng)度呈正相關(guān)，說明Cx43表達(dá)量的改變與平滑肌收縮存在關(guān)系，具體機(jī)制需后續(xù)的深入研究；Cx43表達(dá)的增高能夠影響平滑肌細(xì)胞收縮相關(guān)因子在細(xì)胞間的交流，Cx43表達(dá)越豐富，相關(guān)收縮因子的傳輸性就越強(qiáng)；Cx43參與CVS的機(jī)制可能為防治CVS提供新的思路和靶點(diǎn)。
[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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743
本文編號：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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743
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