發(fā)布時(shí)間：2018-04-26 01:31

  本文選題：CD147 + 巨核細(xì)胞��； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2008年碩士論文

【摘要】： 金屬基質(zhì)蛋白酶誘導(dǎo)劑(matrix metalloproteinase inducer)CD147作為一種粘附分子,參與腫瘤、造血等多種病理生理過(guò)程。CD147廣泛表達(dá)于造血及非造血細(xì)胞,包括上皮細(xì)胞,內(nèi)皮細(xì)胞,粒細(xì)胞,以及在白細(xì)胞中也有弱表達(dá)。最近研究表明CD147參與血細(xì)胞的發(fā)育過(guò)程并與基質(zhì)金屬蛋白酶MMP密切相關(guān)�；|(zhì)金屬蛋白酶(extracellular matrix metalloproteinase,MMP)是一族鋅離子依賴性酶。大量研究表明CD147在多種組織中高表達(dá),是通過(guò)誘導(dǎo)分泌MMPs而發(fā)揮作用。MMPs通過(guò)重塑骨髓造血微環(huán)境ECM參與造血生理和病理過(guò)程,如造血、造血干細(xì)胞動(dòng)員和移植、血液腫瘤等。 Coste等報(bào)道:在紅細(xì)胞發(fā)育過(guò)程的不同階段,表達(dá)在其細(xì)胞膜表而的粘附分子的種類和數(shù)量是有差異的,有些粘附分子隨著紅細(xì)胞的脫核而消失。而CD147在紅細(xì)胞發(fā)育的所有階段,包括成熟紅細(xì)胞均表達(dá),用CD147特異性抗體處理可引起紅細(xì)胞在脾臟的破壞和EPO介導(dǎo)的紅細(xì)胞生成。說(shuō)明CD147在紅細(xì)胞的發(fā)育中發(fā)揮重要作用。大量研究表明血小板的分化途徑和紅細(xì)胞是密切相關(guān)的:如紅系和巨核細(xì)胞系細(xì)胞表達(dá)共同的轉(zhuǎn)錄因子;促紅細(xì)胞生成素(Erythropoietin,EPO)是紅細(xì)胞分化的主要調(diào)節(jié)因子,也與巨核細(xì)胞生成有關(guān);而巨核細(xì)胞特異性生長(zhǎng)因子血小板生成素Thrombopoietin (TPO)可增強(qiáng)紅系祖細(xì)胞的增殖;EPO和TPO與他們各自的細(xì)胞表面受體結(jié)合后,激活相同的信號(hào)轉(zhuǎn)導(dǎo)途徑;長(zhǎng)期大劑量使用EPO可引起小鼠血小板減少。而急性血小板的減少則引起血小板生成增加和紅細(xì)胞生成降低;大量的人白血病細(xì)胞系均同時(shí)表達(dá)紅系和巨核細(xì)胞系特異性蛋白標(biāo)志,改變培養(yǎng)條件可使白血病細(xì)胞向紅系或巨核細(xì)胞系分化等。因此我們推測(cè)CD147在血小板的發(fā)育中可能也發(fā)揮重要作用。 我們發(fā)現(xiàn)成熟血小板表面表達(dá)CD147分子并且與血小板的活化狀態(tài)和功能有關(guān)�；罨“錍D147的表達(dá)較靜息血小板增高、MMP分泌活性增強(qiáng)。加入CD147抗體后,血小板達(dá)到最大聚集率時(shí)間延長(zhǎng),即CD147抗體抑制了血小板的聚集功能。血小板是從骨髓成熟的巨核細(xì)胞漿脫落下來(lái)的具有生物活性的小塊胞質(zhì)。由于血小板沒(méi)有細(xì)胞核,并不是一個(gè)完整的細(xì)胞單位,胞漿中有少量RNA,它所表達(dá)的粘附分子和產(chǎn)生的細(xì)胞因子大部分在巨核細(xì)胞階段合成。巨核細(xì)胞作為血小板的前體細(xì)胞,CD147分子在其分化中的作用尚無(wú)報(bào)道,因此我們首先選取巨核細(xì)胞系HEL細(xì)胞作為模型,檢測(cè)了HEL細(xì)胞表面CD147的表達(dá)和MMP的分泌活性,發(fā)現(xiàn)HEL細(xì)胞CD147和MMP表達(dá)水平均很低。為了研究巨核細(xì)胞在分化為血小板的過(guò)程中CD147及MMP的變化,以及CD147和MMP在分化中的作用,我們克隆了CD147基因,構(gòu)建了含CD147基因的慢病毒載體并包裝病毒;感染HEL細(xì)胞,得到了高表達(dá)CD147的巨核細(xì)胞系HEL細(xì)胞,然后用分化誘導(dǎo)劑TPA進(jìn)行誘導(dǎo)分化。使用光學(xué)顯微鏡觀察分化過(guò)程中細(xì)胞形態(tài),應(yīng)用RT-PCR在轉(zhuǎn)錄水平、流式細(xì)胞術(shù)在蛋白水平檢測(cè)CD147和巨核細(xì)胞分化標(biāo)志CD41的表達(dá)情況、應(yīng)用明膠酶譜分析MMPs的表達(dá)與活性,并考察了MMP抑制劑對(duì)分化的影響。結(jié)果表明,巨核細(xì)胞系HEL細(xì)胞經(jīng)TPA誘導(dǎo)后,細(xì)胞形態(tài)和表面標(biāo)志CD41的表達(dá)均發(fā)生了變化。野生型HEL細(xì)胞經(jīng)TPA誘導(dǎo)分化后,流式結(jié)果顯示隨著HEL細(xì)胞分化標(biāo)志CD41的表達(dá)增加,CD147的表達(dá)也增加,同時(shí)明膠酶譜實(shí)驗(yàn)結(jié)果顯示MMP分泌和活性增強(qiáng),而MMP抑制劑抑制了CD41的表達(dá)。慢病毒介導(dǎo)的CD147表達(dá)增加促進(jìn)了TPA誘導(dǎo)的HEL細(xì)胞的分化,CD41表達(dá)增加,MMP抑制劑同樣抑制HEL分化。這說(shuō)明,CD147分子在巨核細(xì)胞分化過(guò)程中表達(dá)逐漸增加,其作用可能與MMP活性相關(guān)。 結(jié)論:1.發(fā)現(xiàn)CD147在血小板表達(dá)并與血小板狀態(tài)聚集功能相關(guān)�；罨“錍D147表達(dá)較靜息血小板表達(dá)高,而血小板的前體巨核細(xì)胞系HEL細(xì)胞CD147的表達(dá)卻低于成熟血小板,MMP的分泌也表現(xiàn)了相似的規(guī)律。2.通過(guò)慢病毒載體感染獲得了高表達(dá)CD147的巨核細(xì)胞系HEL細(xì)胞。采用Western blot方法檢測(cè)CD147蛋白,表明獲取高表達(dá)CD147的巨核細(xì)胞系HEL細(xì)胞,成功建立了實(shí)驗(yàn)細(xì)胞模型。3.我們建立了誘導(dǎo)巨核細(xì)胞系HEL細(xì)胞分化的方法,觀察細(xì)胞形態(tài)和巨核細(xì)胞分化的表面標(biāo)志,結(jié)果表明TPA確實(shí)誘導(dǎo)了巨核細(xì)胞系HEL細(xì)胞的分化。通過(guò)檢測(cè)分化過(guò)程中CD147的表達(dá)和MMP的分泌與活性,發(fā)現(xiàn)CD147在巨核細(xì)胞分化中可能發(fā)揮了作用。加入MMP抑制劑后,分化被抑制,提示CD147的作用可能與MMP密切相關(guān)。其具體機(jī)制仍有待進(jìn)一步研究。 下一步研究計(jì)劃:研究人臍血造血干細(xì)胞分化發(fā)育為血小板過(guò)程中CD147、MMP的表達(dá)情況和作用,進(jìn)一步探討CD147在血小板發(fā)育中的作用及二者的關(guān)系。
[Abstract]:Metal matrix protease inducer (matrix metalloproteinase inducer) CD147, as a adhesion molecule, participates in a variety of pathophysiological processes, such as tumor and hematopoiesis,.CD147 is widely expressed in hematopoiesis and non hematopoietic cells, including epithelial cells, endothelial cells, granulocytes, and weak expressions in white blood cells. Recent studies have shown that CD147 is involved in blood. The process of cell development is closely related to matrix metalloproteinase MMP. Matrix metalloproteinase (extracellular matrix metalloproteinase, MMP) is a family of zinc dependent enzymes. A large number of studies have shown that CD147 is highly expressed in many tissues. It plays a role in the secretion of MMPs by inducing.MMPs to participate in the remodeling of bone marrow hematopoietic microenvironment ECM participation. Hematopoietic physiological and pathological processes, such as hematopoiesis, hematopoietic stem cell mobilization and transplantation, hematological malignancies, and so on.
Coste and other reports: there are different types and numbers of adhesion molecules expressed in the cell membrane surface at different stages of the erythrocyte development, and some of the adhesion molecules disappear with the denucleation of red cells. And CD147 is expressed in all stages of the development of red blood cells, including the mature red cells, and the CD147 specific antibody can cause red blood to cause red blood. The destruction of the spleen and the formation of red blood cells mediated by EPO indicate that CD147 plays an important role in the development of red blood cells. A large number of studies have shown that the differentiation pathways of platelets are closely related to red cells: the red and megakaryocyte cells express the common transcription factors; the erythropoietin (Erythropoietin, EPO) is the red cell division. The major regulatory factor is also associated with megakaryocyte formation, and megakaryocyte specific growth factor thrombopoietin Thrombopoietin (TPO) can enhance the proliferation of erythroid progenitor cells; EPO and TPO activate the same signal transduction pathway after the combination of their respective cell surface receptors; long term large dose use of EPO can cause a small amount of blood in mice. The reduction of platelets and acute thrombocytopenia cause increased platelet formation and erythropoiesis, and a large number of human leukemia cell lines simultaneously express the specific protein markers of the red and megakaryocyte lines, and the change of culture conditions can cause leukemia cells to differentiate into the red or megakaryocyte lines. Therefore, we speculate that CD147 is in the platelets. It may also play an important role in development.
We found that the expression of CD147 molecules on the surface of mature platelets is related to the activation state and function of platelets. The expression of activated platelet CD147 is higher than that of resting platelets, and the activity of MMP secretion is enhanced. After adding CD147 antibody, the maximum aggregation rate of platelet is prolonged, that is, CD147 antibody inhibits platelet aggregation function. Platelets are A small cell with biological activity from the megakaryocyte pulp that is mature in the bone marrow. Because the platelets have no nuclei, they are not a complete cell unit and a small amount of RNA in the cytoplasm. The adhesion molecules and the resulting cytokines are mostly synthesized in the megakaryocyte stage. Megakaryocyte is the precursor of the platelets. The role of CD147 molecules in its differentiation has not yet been reported. Therefore, we first selected megakaryocyte line HEL cells as a model to detect the expression of CD147 on the surface of HEL cells and the secretory activity of MMP, and found that the expression level of CD147 and MMP in HEL cells was very low. In order to study the changes of CD147 and MMP in the process of megakaryocyte differentiation into platelets, As well as the role of CD147 and MMP in the differentiation, we cloned the CD147 gene, constructed the lentivirus carrier containing the CD147 gene and packaged the virus, infected the HEL cells, and obtained the megakaryocyte HEL cells with high expression of CD147, and then used the differentiation inducer TPA to induce differentiation. The morphology of the cells during the differentiation process was observed with the light microscope, and RT was used to use RT. -PCR at the transcriptional level, flow cytometry at the protein level to detect the expression of CD147 and megakaryocyte differentiation marker CD41, the expression and activity of MMPs were analyzed by gelatinase spectrum, and the effect of MMP inhibitor on the differentiation was investigated. The results showed that the expression of cell morphology and surface marker CD41 in megakaryocyte line HEL cells was induced by TPA. There was a change. After TPA induced differentiation of wild type HEL cells, the flow pattern showed that the expression of CD147 increased with the increase of the expression of HEL cell differentiation marker CD41. Meanwhile, the results of gelase spectrum experiment showed that the secretion and activity of MMP were enhanced, while the MMP inhibitor inhibited the expression of CD41. The increase of CD147 expression promoted the HE of TPA induced HE. The differentiation of L cells, the increase of CD41 expression, and the inhibition of HEL differentiation by MMP inhibitors also suggest that the expression of CD147 molecules is gradually increased during the differentiation of megakaryocyte, and its effect may be related to the activity of MMP.
Conclusion: 1. the expression of CD147 in platelets was found to be related to platelet aggregation. The expression of activated platelets CD147 expression is higher than that of resting platelets, while the expression of CD147 in the HEL cells of the megakaryocyte line of the platelets is lower than that of the mature platelets, and the secretion of MMP is also similar to that of.2. through the infection of the lentivirus carrier. The megakaryocyte HEL cells of CD147 were expressed. The CD147 protein was detected by Western blot method. It showed that the megakaryocyte HEL cells with high expression of CD147 were obtained. The experimental cell model.3. was successfully established. We established the method of inducing the differentiation of the megakaryocyte HEL cell line, and observed the surface markers of the cell form and the megakaryocyte differentiation. The results showed that the cell differentiation of the megakaryocyte lines was marked. TPA did induce the differentiation of the megakaryocyte line HEL cells. By detecting the expression of CD147 and the secretion and activity of MMP during the differentiation, it was found that CD147 may play a role in the differentiation of megakaryocytes. The differentiation was inhibited after adding MMP inhibitors, suggesting that the role of CD147 may be related to the dense cutting of MMP. The specific mechanism of which is still to be further studied.
The next step of the study is to study the expression and role of CD147, MMP in the differentiation and development of human umbilical cord blood hematopoietic stem cells in the process of platelets, and further explore the role of CD147 in the development of platelets and the relationship between the two.

【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

【共引文獻(xiàn)】

相關(guān)期刊論文 前8條

1 劉鵬飛,唐文淵,朱政鳴;CD147和MMP-2、MMP-9與膠質(zhì)細(xì)胞瘤侵襲性的關(guān)系[J];重慶醫(yī)學(xué);2005年11期

2 田福亮;鮑倩;唐志鵬;王書(shū)奎;;CD147在腫瘤血管生成中的作用[J];國(guó)際檢驗(yàn)醫(yī)學(xué)雜志;2011年02期

3 李吉;CD147及其與腫瘤關(guān)系的研究進(jìn)展[J];國(guó)外醫(yī)學(xué)(腫瘤學(xué)分冊(cè));2003年04期

4 李浩,游潮;CD147/EMMPRIN與腦膠質(zhì)瘤關(guān)系的研究進(jìn)展[J];華西醫(yī)學(xué);2005年02期

5 何慧嬋;鐘惟德;;腫瘤中CD147誘導(dǎo)MMPs的分子機(jī)制[J];現(xiàn)代泌尿生殖腫瘤雜志;2009年02期

6 黃靈芝;王字玲;;CD147的研究進(jìn)展[J];生物技術(shù)通訊;2007年03期

7 李存孝,范清宇;CD147結(jié)構(gòu)和功能及其與腫瘤關(guān)系的研究進(jìn)展[J];現(xiàn)代腫瘤醫(yī)學(xué);2005年03期

8 聶桂麗;王艷;甘萍;;CD147與惡性腫瘤關(guān)系的研究進(jìn)展[J];實(shí)驗(yàn)室科學(xué);2009年04期

相關(guān)博士學(xué)位論文 前10條

1 李存孝;CD147在人骨肉瘤細(xì)胞中的表達(dá)及其反義RNA對(duì)骨肉瘤侵襲和轉(zhuǎn)移的影響[D];第四軍醫(yī)大學(xué);2005年

2 郭永川;Ki67核抗原、MMP-2（基質(zhì)金屬蛋白酶-2）、TIMP-2（基質(zhì)金屬蛋白酶-2抑制因子）、CD147、LN-R（層粘連蛋白受體）的表達(dá)與侵襲性垂體腺瘤的關(guān)系[D];吉林大學(xué);2005年

3 霍鋼;侵襲性垂體腺瘤的影像學(xué)與實(shí)驗(yàn)研究[D];重慶醫(yī)科大學(xué);2006年

4 封青川;人cd147的克隆、表達(dá)、純化以其功能研究[D];中國(guó)醫(yī)科大學(xué);2006年

5 李浩;CD147、MMP-2在人腦膠質(zhì)瘤中的表達(dá)及CD147單抗抑制U251細(xì)胞侵襲性的實(shí)驗(yàn)研究[D];四川大學(xué);2007年

6 黨懿;CD147在急性心肌梗死大鼠中的表達(dá)及其與基質(zhì)金屬蛋白酶-9關(guān)系的研究[D];河北醫(yī)科大學(xué);2008年

7 徐靜;腫瘤新靶點(diǎn)HAb18G/CD147與癌—基質(zhì)的交互作用[D];第四軍醫(yī)大學(xué);2008年

8 劉愛(ài)國(guó);CD147及其抑制劑對(duì)非霍奇金淋巴瘤侵襲力影響的臨床及實(shí)驗(yàn)研究[D];華中科技大學(xué);2008年

9 文海榮;CD147對(duì)高糖培養(yǎng)下RPE細(xì)胞生物學(xué)行為影響及衣霉素對(duì)CD147抑制作用研究[D];吉林大學(xué);2010年

10 薛義軍;腺病毒介導(dǎo)的RNAi沉默CD147基因?qū)-24膀胱癌細(xì)胞生物學(xué)行為的影響[D];中國(guó)醫(yī)科大學(xué);2010年

相關(guān)碩士學(xué)位論文 前10條

1 胡杰;EMMPRIN在人肝癌細(xì)胞SMMC-7721中對(duì)多藥耐藥的調(diào)節(jié)作用[D];山西醫(yī)科大學(xué);2011年

2 殷悅;CD147和MMP-9的表達(dá)與涎腺腫瘤侵襲性的關(guān)系[D];吉林大學(xué);2011年

3 周嬌;CD147、MMP-2在口腔鱗癌中的表達(dá)及意義[D];吉林大學(xué);2011年

4 朱嬋;RNAi沉默CD147基因?qū)θ撕戆┘?xì)胞增殖、侵襲、順鉑敏感性及體內(nèi)致瘤性的影響[D];南京師范大學(xué);2011年

5 梁宇翔;CD147和VEGF在終末期腎細(xì)胞癌中的表達(dá)及意義[D];廣州醫(yī)學(xué)院;2010年

6 侯向華;CD147在卵巢癌中的表達(dá)調(diào)節(jié)作用及人卵巢癌原位移植裸鼠模型的構(gòu)建[D];第四軍醫(yī)大學(xué);2004年

7 鄒偉;RNAi沉默CD147基因?qū)β殉舶┘?xì)胞HO-8910pm生物學(xué)行為的影響[D];第四軍醫(yī)大學(xué);2005年

8 田加坤;MMP-2、TIMP-2及CD147與垂體腺瘤侵襲性關(guān)系的研究[D];重慶醫(yī)科大學(xué);2005年

9 楊延冬;原癌基因c-Mct和分子CD147在子宮內(nèi)膜癌中的表達(dá)及其臨床意義[D];青島大學(xué);2006年

10 楊國(guó)康;CD147及LN-R在垂體腺瘤中的表達(dá)及與侵襲性的關(guān)系[D];吉林大學(xué);2006年

，

本文編號(hào)：1803899