本文選題：胎鼠 + 肺泡Ⅱ型上皮細(xì)胞�。� 參考：《重慶醫(yī)科大學(xué)》2009年博士論文

【摘要】： 第一部分早產(chǎn)胎鼠肺泡II型上皮細(xì)胞分離、純化、培養(yǎng)及鑒定 目的 探討胎鼠肺泡Ⅱ型上皮細(xì)胞(AECⅡ)的分離、純化及原代培養(yǎng)方法,建立胎鼠AECⅡ細(xì)胞模型,為有關(guān)胎兒肺發(fā)育及新生兒肺部疾病的研究奠定基礎(chǔ)。 方法 采用胰酶結(jié)合膠原酶的消化方法,分離肺組織細(xì)胞成份,然后經(jīng)差速離心和差速貼壁的方法純化AECⅡ,進(jìn)行原代培養(yǎng);通過臺盼藍(lán)染色檢測細(xì)胞活力,倒置相差顯微鏡觀察細(xì)胞生長特點(diǎn)及形態(tài)特征,透射電鏡鑒定,改良巴氏染色檢測細(xì)胞純度以及免疫熒光技術(shù)檢測表面蛋白C(SP-C)的表達(dá)。 結(jié)果 每3～5只胎鼠可獲得AECⅡ(36±5)×106,活力(98±2)%。鏡下觀察原代AECⅡ呈島狀生長,外觀呈多邊形或立方形。透射電鏡可見特征性的板層小體,改良巴氏染色見胞質(zhì)內(nèi)有較多顆粒,純度為96±3%,呈現(xiàn)SP-C綠色免疫熒光的細(xì)胞占96%以上。 結(jié)論 利用胰酶和膠原酶消化,以及差速離心和差速貼壁的方法可成功分離出高產(chǎn)量、高純度的胎鼠AECⅡ,能滿足體外進(jìn)一步實(shí)驗(yàn)的需要。 第二部分高氧暴露對早產(chǎn)鼠肺泡Ⅱ型上皮細(xì)胞的氧化損傷及CGRP的保護(hù)作用 目的 觀察高氧對早產(chǎn)鼠肺泡Ⅱ型上皮細(xì)胞(AECⅡ)的影響以及降鈣素基因相關(guān)肽(CGRP)對AECⅡ的保護(hù)作用。 方法 將原代分離培養(yǎng)的孕19d早產(chǎn)鼠AECⅡ接種至6孔培養(yǎng)板,實(shí)驗(yàn)隨機(jī)分為空氣組、高氧組、高氧CGRP組、高氧CGRP受體拮抗劑組�？諝饨M和高氧組分別置于體積分?jǐn)?shù)為21%的空氣和60%的氧氣中暴露24h;高氧CGRP組在暴露前加入CGRP;高氧CGRP拮抗劑組在高氧CGRP組基礎(chǔ)上加入CGRP受體拮抗劑(CGRP8-37)。培養(yǎng)24h后,用分光光度計(jì)測定各組丙二醛(MDA)、總抗氧化能力(TAOC)、超氧化物歧化酶(SOD)水平;用流式細(xì)胞儀檢測活性氧(ROS)和細(xì)胞凋亡率;用逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)測定表面活性蛋白C (SP-C)的mRNA表達(dá)。 結(jié)果 與空氣組比較,高氧組MDA、ROS及細(xì)胞調(diào)亡率均顯著增高,TAOC、SOD水平及SP-C mRNA表達(dá)均顯著降低(P均0.01)。與高氧組比較,高氧CGRP組細(xì)胞MDA、ROS水平及細(xì)胞調(diào)亡率均顯著下降;而TAOC、SOD水平及SP-C mRNA表達(dá)均明顯增高(P0.01)。高氧CGRP拮抗劑組與高氧組各指標(biāo)比較差異均無統(tǒng)計(jì)學(xué)意義。 結(jié)論 60%氧暴露24h可導(dǎo)致早產(chǎn)鼠AECⅡ發(fā)生氧化損傷,誘導(dǎo)細(xì)胞凋亡及SP-C mRNA表達(dá)下降;而CGRP可部分減輕AECⅡ的氧化損傷,減少凋亡,促進(jìn)SP-CmRNA表達(dá),對高氧損傷的AECⅡ起保護(hù)作用。 第三部分CGRP對60%氧暴露早產(chǎn)鼠AECⅡ生長增殖的影響 目的 探討降鈣素基因相關(guān)肽(CGRP)對60%氧暴露早產(chǎn)鼠肺泡Ⅱ型上皮細(xì)胞(AECⅡ)生長增殖的影響。 方法 原代分離培養(yǎng)孕19d早產(chǎn)鼠AECⅡ,隨機(jī)分為6組:空氣組、空氣CGRP組、空氣CGRP拮抗劑組、高氧組、高氧CGRP組、高氧CGRP拮抗劑組�？諝饨M和高氧組分別在氧體積分?jǐn)?shù)為21%的空氣和60%的氧氣中暴露24h;空氣或高氧CGRP組在置于空氣或高氧環(huán)境前加入CGRP;空氣或高氧CGRP拮抗劑組同時(shí)加入CGRP和CGRP受體拮抗劑后,再置于空氣或60%的氧氣中培養(yǎng)24h。先用MTT比色法測定不同濃度CGRP(10-10～10-7M)對正常AECⅡ生長的影響,以確定CGRP的實(shí)驗(yàn)最佳濃度;分別采用MTT法和流式細(xì)胞術(shù)測定各組細(xì)胞增殖能力和細(xì)胞周期,逆轉(zhuǎn)錄聚合酶鏈反應(yīng)和免疫熒光技術(shù)測定表面活性蛋白C (SP-C)的mRNA及蛋白表達(dá)。 結(jié)果 MTT法結(jié)果顯示,CGRP從10-10～10-8M,呈劑量依賴方式促進(jìn)正常培養(yǎng)的早產(chǎn)鼠AECⅡ生長,因此選擇10-8M CGRP來干預(yù)細(xì)胞。加入10-8M CGRP還可使正常培養(yǎng)的AECⅡ進(jìn)入G2/M及S期的比例增多,SP-C mRNA及SP-C蛋白表達(dá)增高(與空氣組相比,P均 0.01)。暴露于60%氧24h后,細(xì)胞存活率下降,G0/G1期細(xì)胞比例增高,G2/M及S期細(xì)胞相應(yīng)降低,SP-C mRNA及SP-C蛋白表達(dá)低下(與空氣組比較,P均0.01)。而預(yù)先加入10-8M CGRP后,促進(jìn)了高氧暴露AECⅡ的增殖能力,使S及G2/M期細(xì)胞增多,并可提高AECⅡ的SP-C mRNA及SP-C蛋白表達(dá)水平(與高氧組及高氧CGRP拮抗劑組比較,P0.01)。 結(jié)論 60%氧暴露24h可抑制早產(chǎn)鼠AECⅡ增殖分化,而CGRP可促進(jìn)AECⅡ生長,部分解除高氧對AECⅡ的抑制作用。 第四部分PKCα信號轉(zhuǎn)導(dǎo)途徑介導(dǎo)了CGRP對高氧肺泡Ⅱ型上皮細(xì)胞損傷的保護(hù)作用 目的 通過測定PKCα和NF-κB的活化情況,從細(xì)胞內(nèi)信號轉(zhuǎn)導(dǎo)通路這一角度探討CGRP對高氧肺泡上皮細(xì)胞損傷的保護(hù)作用機(jī)制。 方法 原代分離培養(yǎng)孕19d早產(chǎn)鼠AECⅡ,隨機(jī)分為6組:空氣組、空氣CGRP組、空氣CGRP拮抗劑組、高氧組、高氧CGRP組、高氧CGRP拮抗劑組�？諝饨M和高氧組分別在氧體積分?jǐn)?shù)為21%的空氣和60%的氧氣中暴露24h;空氣或高氧CGRP組在置于空氣或高氧環(huán)境前加入CGRP,空氣或高氧CGRP拮抗劑組同時(shí)加入CGRP和CGRP受體拮抗劑,再置于空氣或60%的氧氣中培養(yǎng)24h。用Western blot檢測胞膜和胞漿PKCα的表達(dá)變化,激光共聚焦檢測NF-κB的核表達(dá)情況。 結(jié)果 在正常培養(yǎng)的AECⅡ細(xì)胞中加入CGRP后,胞膜與胞漿PKCα的比值顯著增高,NFκB的熒光較強(qiáng),與空氣組及空氣CGRP拮抗劑組相比有顯著性差異(P0.01)。細(xì)胞暴露于高氧后,胞膜與胞漿PKC比值顯著低于空氣組,而NFκB的熒光強(qiáng)度高于空氣組(p0.01)。高氧CGRP組二者比值及核內(nèi)NFκB熒光強(qiáng)度高于高氧組及高氧CGRP拮抗劑組,差異有顯著性意義(p0.01)。 結(jié)論 PKCα介導(dǎo)了CGRP對細(xì)胞的信號傳遞過程,參與了CGRP對高氧AECⅡ損傷的保護(hù)作用,而NF-κB是PKCα的下游信號,部分執(zhí)行了PKCα傳遞的保護(hù)功能
[Abstract]:Part 1 isolation, purification, culture and identification of alveolar type II epithelial cells from preterm fetal rats
objective
To explore the isolation, purification and primary culture of type II epithelial cells (AEC II) of fetal rat alveolar type II epithelial cells (AEC II), and to establish a model of AEC II cells in fetal mice, which lay the foundation for the study of fetal lung development and neonatal pulmonary disease.
Method
The cell components of lung tissue were separated by trypsin and collagenase digestion. Then AEC II was purified by differential centrifugation and differential adherence to primary culture. Cell viability was detected by trypan blue staining, and cell growth characteristics and morphological characteristics were observed by inverted phase contrast microscope. Transmission electron microscopy was used to identify cells and improve the detection of cells by PAP staining. Purity and immunofluorescence technique were used to detect the expression of surface protein C (SP-C).
Result
AEC II (36 + 5) * 106 and vitality (98 + 2) were obtained in every 3~5 fetal mice. The original AEC II was observed under the microscope. The appearance was polygonal or cubic. The transmission electron microscope showed the characteristic lamellar body. The improved PAP staining showed that there were more granules in the cytoplasm, the purity was 96 + 3%, and the cells showing SP-C green immunofluorescence were more than 96%.
conclusion
The use of trypsin and collagenase digestion, differential centrifugation and differential adherence can successfully isolate AEC II of high yield and high purity fetal mice, which can meet the needs of further experiments in vitro.
The second part is the oxidative damage of alveolar type II epithelial cells induced by hyperoxia exposure in premature rats and the protective effect of CGRP.
objective
Objective To observe the effect of hyperoxia on alveolar type II epithelial cells (AEC II) in premature rats and the protective effect of calcitonin gene-related peptide (CGRP) on AEC II.
Method
The pregnant 19d preterm rat AEC II was inoculated to 6 Hole culture plate. The experiment was randomly divided into air group, hyperoxia group, hyperoxic CGRP group and hyperoxic CGRP receptor antagonist group. Air group and hyperoxic group were exposed to 24h in the air of volume fraction of 21% and 60% of oxygen in 60% oxygen; high oxygen CGRP group was added to CGRP before exposure; the group of high oxygen CGRP antagonist was in the group The CGRP receptor antagonist (CGRP8-37) was added to the hyperoxic CGRP group. After the culture of 24h, a spectrophotometer was used to determine all groups of malondialdehyde (MDA), the total antioxidant capacity (TAOC), the level of superoxide dismutase (SOD), the detection of reactive oxygen species (ROS) and the apoptosis rate by flow cytometry, and the determination of the surface active protein C (SP-) by reverse transcription polymerase chain reaction (RT-PCR). MRNA expression of C).
Result
Compared with the air group, the MDA, ROS and cell apoptosis rate of hyperoxia group were significantly increased, TAOC, SOD level and SP-C mRNA expression were significantly decreased (P 0.01). Compared with the hyperoxia group, the MDA, ROS level and cell apoptosis rate of the hyperoxic CGRP group decreased significantly, while TAOC, SOD level and higher expression were higher. There was no significant difference in each index of oxygen group.
conclusion
60% oxygen exposure to 24h can lead to oxidative damage of AEC II in premature rats, induce apoptosis and decrease the expression of SP-C mRNA, and CGRP can partially alleviate the oxidative damage of AEC II, decrease apoptosis, promote the expression of SP-CmRNA, and protect the AEC II injured by hyperoxia.
The third part is the effect of CGRP on the growth and proliferation of AEC II in premature rats exposed to 60% oxygen.
objective
Objective to investigate the effects of calcitonin gene-related peptide (CGRP) on the growth and proliferation of alveolar type II epithelial cells (AEC II) in 60% oxygen exposed premature rats.
Method
AEC II of preterm 19d preterm rats was randomly divided into 6 groups: air group, air CGRP group, air CGRP antagonist group, hyperoxia group, hyperoxic CGRP group, hyperoxic CGRP antagonist group. Air group and hyperoxic group exposed 24h in oxygen volume fraction of 21% air and 60% oxygen respectively; air or hyperoxic CGRP group was added before air or high oxygen environment. After entering CGRP, air or hyperoxic CGRP antagonist group was added to CGRP and CGRP receptor antagonists, and then cultured in air or 60% of oxygen, 24h. was first used to determine the effect of CGRP (10-10 ~ 10-7M) on normal AEC II growth by MTT colorimetry, in order to determine the optimal concentration of CGRP, and the cells were determined by MTT method and flow cytometry respectively. Proliferative capacity and cell cycle, reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence technique were used to detect the expression of mRNA and protein of surface active protein C (SP-C).
Result
The results of MTT showed that CGRP from 10-10 to 10-8M promoted the growth of AEC II in normal cultured preterm rats, so 10-8M CGRP was selected to interfere with the cells. The proportion of AEC II into G2/M and S phase in normal culture was increased by adding 10-8M CGRP, and increased (0.01) from the air group (compared with the air group). 60% After oxygen 24h, the cell survival rate decreased, the proportion of G0/G1 cells increased, G2/M and S cells decreased correspondingly, SP-C mRNA and SP-C protein were low (P was 0.01). The proliferation of AEC II was promoted after the addition of 10-8M CGRP. Reached the level (compared with hyperoxia group and hyperoxia CGRP antagonist group, P0.01).
conclusion
60% oxygen exposure 24h can inhibit the proliferation and differentiation of AEC II in premature rats, while CGRP can promote AEC II growth, partially relieving the inhibitory effect of hyperoxia on AEC II.
In the fourth part, PKC alpha signal transduction mediates the protective effect of CGRP on hyperoxia alveolar type II epithelial cells.
objective
By measuring the activation of PKC and NF- kappa B, we explored the protective mechanism of CGRP on the injury of hyperoxia alveolar epithelial cells from the signal transduction pathway.
Method
AEC II of preterm 19d preterm rats was randomly divided into 6 groups: air group, air CGRP group, air CGRP antagonist group, hyperoxia group, hyperoxic CGRP group, hyperoxic CGRP antagonist group. Air group and hyperoxic group exposed 24h in oxygen volume fraction of 21% air and 60% oxygen respectively; air or hyperoxic CGRP group was added before air or high oxygen environment. In CGRP, air or hyperoxic CGRP antagonist group was added to CGRP and CGRP receptor antagonists, and then in air or 60% oxygen, 24h. was cultured with Western blot to detect the expression of PKC alpha in cell membrane and cytoplasm, and the nuclear expression of NF- kappa B was detected by confocal laser.
Result
After adding CGRP to normal AEC II cells, the ratio of cell membrane to cytoplasmic PKC a was significantly higher, and the fluorescence of NF kappa B was stronger than that in air group and air CGRP antagonist group (P0.01). After exposure to hyperoxia, the ratio of cell membrane to cytoplasmic PKC was significantly lower than that in air group, and the fluorescence intensity of NF kappa B was higher than that of air group (P0.01). The ratio of the two groups and the fluorescence intensity of NF kappa B in the oxygen CGRP group were higher than those in the hyperoxia group and the hyperoxia CGRP antagonist group (P0.01).
conclusion
PKC - alpha mediates the signaling process of CGRP to cells, and participates in the protective effect of CGRP on hyperoxia AEC II damage, and NF- - kappa B is a downstream signal of PKC alpha, partially performing the protection function of PKC alpha transmission
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R363

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