表達(dá)雙報(bào)告基因的慢病毒顆粒的制備及體外標(biāo)記小鼠間充質(zhì)干細(xì)胞
發(fā)布時(shí)間：2018-07-27 16:36
【摘要】：間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs)是一類具有高度自我更新和多向分化潛能的成體干細(xì)胞,由于取材方便、體外能大量擴(kuò)增,近年成為干細(xì)胞領(lǐng)域的研究熱點(diǎn)之一。MSCs主要來(lái)源于骨髓和脂肪組織,其次也可從臍血、外周血、胎盤、肺、羊水、肌肉、網(wǎng)膜等組織獲得。它能夠向血管、心肌、骨、脂肪、神經(jīng)和皮膚等多個(gè)胚層組織分化,是組織工程的理想種子細(xì)胞。在各種原因所致的嚴(yán)重創(chuàng)傷下許多受損的組織和臟器需要及時(shí)的修復(fù)和替代,間充質(zhì)干細(xì)胞的可塑性使其有可能成為治療多種疾病的有力手段。 MSCs的免疫原性低,不表達(dá)MHC-Ⅱ類分子和CD80、CD86等協(xié)同刺激分子。研究表明,MSCs能夠提供細(xì)胞因子和生長(zhǎng)因子支持造血干細(xì)胞的擴(kuò)增和成熟。另外,它能夠抑制T細(xì)胞增殖和免疫細(xì)胞因子的產(chǎn)生,具有免疫調(diào)節(jié)作用,能夠誘導(dǎo)免疫耐受,可用于造血干細(xì)胞移植和自身免疫性疾病的治療。 本研究應(yīng)用膠原酶消化骨片的方法成功分離小鼠MSCs,并在體外進(jìn)行培養(yǎng)。這種方法獲得的細(xì)胞有均一的表型,不表達(dá)造血和內(nèi)皮的標(biāo)記分子CD11b、CD31、CD45、CD34,表達(dá)細(xì)胞粘附分子CD29、CD44、CD105和干細(xì)胞標(biāo)志Sca-1。其具有貼壁性,能夠向脂肪、成骨和軟骨誘導(dǎo)分化,符合MSCs的鑒定標(biāo)準(zhǔn)。 體外穩(wěn)定高效的標(biāo)記細(xì)胞,對(duì)研究其在體內(nèi)的分布、遷移、增殖及分化有重要意義。在傳統(tǒng)的動(dòng)物實(shí)驗(yàn)中,往往需要處死大批動(dòng)物后才能獲得實(shí)驗(yàn)結(jié)果,很難直觀、活體、動(dòng)態(tài)地連續(xù)觀察細(xì)胞的分布遷移情況,限制了對(duì)細(xì)胞,尤其是早期細(xì)胞的分布、遷移、增殖、分化、持續(xù)時(shí)間和治療效果的研究和評(píng)價(jià)。因此,建立便于檢測(cè)的細(xì)胞標(biāo)記方法具有非常重要的意義。目前,實(shí)驗(yàn)室研究常用的一些體外細(xì)胞標(biāo)記方法有熒光染料標(biāo)記方法、熒光報(bào)告基團(tuán)及生物發(fā)光方法和放射性核素標(biāo)記法等。紅色熒光蛋白(red fluorescent protein,RFP)/螢火蟲熒光素酶雙標(biāo)技術(shù)(firefly luciferase,Fluc)就是將化學(xué)發(fā)光和熒光成像技術(shù)結(jié)合起來(lái),化學(xué)發(fā)光技術(shù)是用熒光素酶(Luciferase)基因標(biāo)記細(xì)胞或DNA,而熒光技術(shù)則采用熒光報(bào)告基團(tuán)(GFP、RFP,Cyt及Dyes等)進(jìn)行標(biāo)記,然后利用一套非常靈敏的光學(xué)檢測(cè)儀器,直接監(jiān)控活體生物體內(nèi)的細(xì)胞活動(dòng)和基因行為。該技術(shù)在干細(xì)胞學(xué)研究領(lǐng)域中的應(yīng)用,迄今在國(guó)內(nèi)的報(bào)道還為數(shù)不多,其不僅繼承了RFP或Fluc單標(biāo)技術(shù)的優(yōu)勢(shì),而且突破了后者的局限性,結(jié)合活體成像技術(shù),可以直接監(jiān)測(cè)干細(xì)胞在動(dòng)物體內(nèi)的生物學(xué)行為,是研究細(xì)胞輸注后在實(shí)驗(yàn)動(dòng)物體內(nèi)分布和分化的強(qiáng)有力手段,在醫(yī)學(xué)和生物醫(yī)學(xué)研究領(lǐng)域中具有越來(lái)越廣泛的應(yīng)用前景。慢病毒系統(tǒng)能夠有效地感染各類細(xì)胞,實(shí)現(xiàn)外源基因在細(xì)胞內(nèi)的穩(wěn)定表達(dá),對(duì)雙報(bào)告基因在細(xì)胞中的表達(dá)具有重要意義。 本研究通過(guò)構(gòu)建螢火蟲熒光素酶和紅色熒光蛋白雙報(bào)告基因表達(dá)的慢病毒載體,轉(zhuǎn)染293T細(xì)胞后,純化獲得具有高滴度和感染活性的慢病毒顆粒,轉(zhuǎn)導(dǎo)特定細(xì)胞系(Hela),采用熒光顯微鏡和熒光素酶報(bào)告基因檢測(cè)試劑盒檢測(cè)RFP和Fluc雙報(bào)告基因的表達(dá)。采用酶切和測(cè)序的方法證實(shí)rfp和fluc基因成功連入目的載體,所構(gòu)建pLenti-Fluc-RFP慢病毒重組質(zhì)粒序列符合預(yù)期。獲得的慢病毒滴度測(cè)定為1×107TU/ml。將病毒轉(zhuǎn)導(dǎo)Hela細(xì)胞系后,熒光顯微鏡下可見大量RFP表達(dá)陽(yáng)性細(xì)胞,熒光發(fā)光檢測(cè)儀也能通過(guò)檢測(cè)發(fā)光單位證實(shí)轉(zhuǎn)導(dǎo)細(xì)胞具有較高的熒光素酶活性。慢病毒載體的成功構(gòu)建為研究間充質(zhì)干細(xì)胞在體內(nèi)的動(dòng)態(tài)分布奠定了基礎(chǔ)。 在再生醫(yī)學(xué)及組織工程中MSCs雖然得到了廣泛應(yīng)用,但其在臨床應(yīng)用中的安全性仍不是很清楚。目前有許多關(guān)于MSCs與腫瘤關(guān)系的研究,其能否轉(zhuǎn)化成惡性腫瘤細(xì)胞及在體內(nèi)外對(duì)腫瘤細(xì)胞增殖的影響,各研究結(jié)果不同。而且目前關(guān)于MSCs的許多生物學(xué)特性及分子調(diào)控機(jī)制尚不十分清楚,對(duì)其體內(nèi)分布、歸巢、增殖和定植的研究尚處于初級(jí)階段,有待進(jìn)一步探索。MSCs移植的方法和最佳時(shí)機(jī)等還不確定。經(jīng)體外分離、培養(yǎng)的干細(xì)胞是一類具有多向分化能力的細(xì)胞,其是否存在基因突變,植入機(jī)體后是否有癌變的可能,對(duì)于MSCs的橫向分化潛能,也有科學(xué)家提出質(zhì)疑。由于目前所進(jìn)行的臨床研究較少,病例數(shù)較少,其有效性和安全性等問(wèn)題仍有待進(jìn)一步觀察。目前已有的研究工作表明循環(huán)的骨髓間充質(zhì)干細(xì)胞可以遷延到不同的部位,如骨髓、肺、肝、脾、心臟、腦部、胰島等;經(jīng)靜脈注射后骨髓間充質(zhì)干細(xì)胞最初的分布器官是肺、肝、腎,特別在注射后第一天就發(fā)現(xiàn)骨髓間充質(zhì)干細(xì)胞大部分滯留于肺泡,而注射七天后才發(fā)現(xiàn)在肌肉、心臟、腦和脾的分布;在機(jī)體組織受損傷時(shí),骨髓MSCs可經(jīng)骨髓動(dòng)員自發(fā)到達(dá)損傷部位,并在局部微環(huán)境誘導(dǎo)下分化為特異的組織細(xì)胞參與自身修復(fù);動(dòng)員循環(huán)中的骨髓間充質(zhì)干細(xì)胞遷延到特定組織或器官的相關(guān)機(jī)制尚沒(méi)有統(tǒng)一的說(shuō)法,但一般認(rèn)為涉及多種分子及信號(hào)通路的相互作用。 本研究應(yīng)用制備的表達(dá)雙報(bào)告基因系統(tǒng)的慢病毒顆粒體外標(biāo)記感染小鼠MSCs,經(jīng)檢測(cè)感染后的MSCs能夠穩(wěn)定表達(dá)RFP和Fluc報(bào)告基因,且形態(tài)均一,呈成纖維樣生長(zhǎng),通過(guò)細(xì)胞表型和體外誘導(dǎo)分化鑒定證實(shí)細(xì)胞一般生物學(xué)特性未受病毒影響,同時(shí)結(jié)合流式細(xì)胞分選技術(shù)使RFP/Fluc雙標(biāo)表達(dá)率接近100%,為后期體外大量擴(kuò)增培養(yǎng)細(xì)胞,采用活體成像技術(shù)監(jiān)測(cè)穩(wěn)定感染細(xì)胞在小鼠體內(nèi)深部組織的分布情況的實(shí)驗(yàn)奠定基礎(chǔ)。
[Abstract]:Mesenchymal stem cells (MSCs) is a kind of adult stem cells with high self-renewal and pluripotent differentiation potential. Because of the convenience of taking materials, it can be expanded in large quantities in vitro. In recent years,.MSCs has become one of the hot spots in the field of stem cells..MSCs is mainly derived from bone marrow and fat tissue. Secondly, it can also be derived from umbilical blood, peripheral blood, placenta, lung, sheep. Water, muscle, omentum and other tissues are obtained. It can differentiate into blood vessels, myocardium, bone, fat, nerve and skin. It is the ideal seed cell for tissue engineering. Under the severe trauma caused by various causes, many damaged tissues and organs need to be repaired and replaced in time. The plasticity of mesenchymal stem cells may make it possible. A powerful means to treat a variety of diseases.
MSCs has low immunogenicity, does not express MHC- class II molecules and CD80, CD86 and other synergistic stimulators. The study shows that MSCs can provide cytokines and growth factors to support the expansion and maturation of hematopoietic stem cells. In addition, it can inhibit the proliferation of T cells and the production of immune cell factors, which can induce immune regulation and can induce immune tolerance. It is used for hematopoietic stem cell transplantation and autoimmune diseases.
This study used collagenase to digest bone slices successfully and successfully isolated MSCs in mice and cultured in vitro. This method has a homogeneous phenotype that does not express CD11b, CD31, CD45, CD34 of hematopoietic and endothelial markers, CD29, CD44, CD105, and stem cell markers, which are adhered to the adherence of CD44, CD105 and stem cells, and can be directed to fat. Osteogenesis and cartilage differentiation were in accordance with the identification criteria of MSCs.
Stable and efficient labelled cells in vitro are of great significance for studying their distribution, migration, proliferation and differentiation in the body. In traditional animal experiments, it is often necessary to kill a large number of animals to obtain experimental results. It is difficult to visualized, live, dynamically and continuously observe the distribution and migration of the cells, and limit the cells, especially the early cells. Research and evaluation of distribution, migration, proliferation, differentiation, duration and therapeutic effect. Therefore, it is of great significance to establish a method for detecting cell markers. At present, some methods commonly used in laboratory study are fluorescent dye labeling, fluorescence report groups and bioluminescence methods and radionuclides. Red fluorescent protein (RFP) / fluorescein double standard (firefly luciferase, Fluc) is a combination of chemiluminescence and fluorescence imaging, and chemiluminescence is used to mark cell or DNA with luciferase (Luciferase) gene, while fluorescence technology uses fluorescence report group (GFP, RFP, C). YT and Dyes, etc., are labeled, and then use a very sensitive optical instrument to directly monitor cell activity and gene behavior in living organisms. The application of this technology in the field of dry cytology has not been reported so far in China, not only inheriting the advantages of RFP or Fluc single standard technology, but also breaking through the latter The limitation, combined with living imaging technology, can directly monitor the biological behavior of stem cells in the animal body. It is a powerful means to study the distribution and differentiation of the cells after the cell infusion in the experimental animals. It has a more and more extensive application prospect in the field of medical and biomedical research. The slow virus system can effectively infect various kinds of fine. The stable expression of foreign genes in cells is of great significance to the expression of double reporter genes in cells.
In this study, the lentivirus vector expressed by fluorescein and red fluorescent protein gene was constructed. After transfecting 293T cells, the lentivirus particles with high titer and infection activity were purified, and the specific cell line (Hela) was transduced. The double reports of RFP and Fluc were detected by fluorescence microscopy and fluoro enzyme reporter gene detection kit. RFP and fluc genes were successfully linked to the target carrier by enzyme digestion and sequencing, and the sequence of the recombinant plasmid of pLenti-Fluc-RFP lentivirus was expected. The acquired lentivirus titer was 1 * 107TU/ml. and a large number of RFP positive cells were found under fluorescence microscopy. The light detector can also prove that the transduced cells have high luciferase activity by detecting the luminescent units. The successful construction of the lentivirus vector has laid the foundation for the study of the dynamic distribution of mesenchymal stem cells in the body.
Although MSCs has been widely used in regenerative medicine and tissue engineering, the safety of its clinical application is still not very clear. There are many studies on the relationship between MSCs and tumor, whether it can be transformed into malignant tumor cells and the effects on the proliferation of tumor cells in vivo and in vivo, and the results are different for MSCs. Many biological characteristics and molecular regulatory mechanisms are still not very clear. The research on its distribution, homing, proliferation and colonization is still in the primary stage. It is still uncertain that the methods and the best time for.MSCs transplantation are still to be further explored. The possibility of canceration after the gene mutation is implanted into the body. There is also a challenge for the lateral differentiation potential of MSCs. The number of cases is less, the number of cases is less, its effectiveness and safety remain to be further observed. To move to different parts, such as bone marrow, lung, liver, spleen, heart, brain, islet, and so on. After intravenous injection, the initial distribution organ of bone marrow mesenchymal stem cells is lung, liver, kidney, and the bone marrow mesenchymal stem cells were found most of the pulmonary alveolus on the first day after injection, and the distribution of muscle, heart, brain and spleen was found seven days after injection. When the body tissue is damaged, bone marrow MSCs can reach the site of injury spontaneously through bone marrow mobilization, and differentiate into specific tissue cells in local microenvironment to participate in self repair. There is no unified statement on the mechanism of mobilizing bone marrow mesenchymal stem cells in the circulation to specific tissues or organs. The interaction of species and signal pathways.
In this study, the lentivirus particles expressed by the prepared double reporter gene system were labeled with MSCs in vitro. After detection of infected MSCs, the expression of RFP and Fluc reporter genes were stable, and the morphology was homogeneous and fibroid growth. The general biological characteristics of the cells were confirmed to be unaffected by the virus by the phenotype and differentiation of the cells in vitro. At the same time, combined with flow cytometry, the expression rate of RFP/Fluc double standard was close to 100%, which was a large number of cultured cells in the later period, and the living body imaging technique was used to monitor the distribution of the stable infected cells in the deep tissues of the mice.
【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】：2011
【分類號(hào)】：R329
本文編號(hào)：2148437
[Abstract]:Mesenchymal stem cells (MSCs) is a kind of adult stem cells with high self-renewal and pluripotent differentiation potential. Because of the convenience of taking materials, it can be expanded in large quantities in vitro. In recent years,.MSCs has become one of the hot spots in the field of stem cells..MSCs is mainly derived from bone marrow and fat tissue. Secondly, it can also be derived from umbilical blood, peripheral blood, placenta, lung, sheep. Water, muscle, omentum and other tissues are obtained. It can differentiate into blood vessels, myocardium, bone, fat, nerve and skin. It is the ideal seed cell for tissue engineering. Under the severe trauma caused by various causes, many damaged tissues and organs need to be repaired and replaced in time. The plasticity of mesenchymal stem cells may make it possible. A powerful means to treat a variety of diseases.
MSCs has low immunogenicity, does not express MHC- class II molecules and CD80, CD86 and other synergistic stimulators. The study shows that MSCs can provide cytokines and growth factors to support the expansion and maturation of hematopoietic stem cells. In addition, it can inhibit the proliferation of T cells and the production of immune cell factors, which can induce immune regulation and can induce immune tolerance. It is used for hematopoietic stem cell transplantation and autoimmune diseases.
This study used collagenase to digest bone slices successfully and successfully isolated MSCs in mice and cultured in vitro. This method has a homogeneous phenotype that does not express CD11b, CD31, CD45, CD34 of hematopoietic and endothelial markers, CD29, CD44, CD105, and stem cell markers, which are adhered to the adherence of CD44, CD105 and stem cells, and can be directed to fat. Osteogenesis and cartilage differentiation were in accordance with the identification criteria of MSCs.
Stable and efficient labelled cells in vitro are of great significance for studying their distribution, migration, proliferation and differentiation in the body. In traditional animal experiments, it is often necessary to kill a large number of animals to obtain experimental results. It is difficult to visualized, live, dynamically and continuously observe the distribution and migration of the cells, and limit the cells, especially the early cells. Research and evaluation of distribution, migration, proliferation, differentiation, duration and therapeutic effect. Therefore, it is of great significance to establish a method for detecting cell markers. At present, some methods commonly used in laboratory study are fluorescent dye labeling, fluorescence report groups and bioluminescence methods and radionuclides. Red fluorescent protein (RFP) / fluorescein double standard (firefly luciferase, Fluc) is a combination of chemiluminescence and fluorescence imaging, and chemiluminescence is used to mark cell or DNA with luciferase (Luciferase) gene, while fluorescence technology uses fluorescence report group (GFP, RFP, C). YT and Dyes, etc., are labeled, and then use a very sensitive optical instrument to directly monitor cell activity and gene behavior in living organisms. The application of this technology in the field of dry cytology has not been reported so far in China, not only inheriting the advantages of RFP or Fluc single standard technology, but also breaking through the latter The limitation, combined with living imaging technology, can directly monitor the biological behavior of stem cells in the animal body. It is a powerful means to study the distribution and differentiation of the cells after the cell infusion in the experimental animals. It has a more and more extensive application prospect in the field of medical and biomedical research. The slow virus system can effectively infect various kinds of fine. The stable expression of foreign genes in cells is of great significance to the expression of double reporter genes in cells.
In this study, the lentivirus vector expressed by fluorescein and red fluorescent protein gene was constructed. After transfecting 293T cells, the lentivirus particles with high titer and infection activity were purified, and the specific cell line (Hela) was transduced. The double reports of RFP and Fluc were detected by fluorescence microscopy and fluoro enzyme reporter gene detection kit. RFP and fluc genes were successfully linked to the target carrier by enzyme digestion and sequencing, and the sequence of the recombinant plasmid of pLenti-Fluc-RFP lentivirus was expected. The acquired lentivirus titer was 1 * 107TU/ml. and a large number of RFP positive cells were found under fluorescence microscopy. The light detector can also prove that the transduced cells have high luciferase activity by detecting the luminescent units. The successful construction of the lentivirus vector has laid the foundation for the study of the dynamic distribution of mesenchymal stem cells in the body.
Although MSCs has been widely used in regenerative medicine and tissue engineering, the safety of its clinical application is still not very clear. There are many studies on the relationship between MSCs and tumor, whether it can be transformed into malignant tumor cells and the effects on the proliferation of tumor cells in vivo and in vivo, and the results are different for MSCs. Many biological characteristics and molecular regulatory mechanisms are still not very clear. The research on its distribution, homing, proliferation and colonization is still in the primary stage. It is still uncertain that the methods and the best time for.MSCs transplantation are still to be further explored. The possibility of canceration after the gene mutation is implanted into the body. There is also a challenge for the lateral differentiation potential of MSCs. The number of cases is less, the number of cases is less, its effectiveness and safety remain to be further observed. To move to different parts, such as bone marrow, lung, liver, spleen, heart, brain, islet, and so on. After intravenous injection, the initial distribution organ of bone marrow mesenchymal stem cells is lung, liver, kidney, and the bone marrow mesenchymal stem cells were found most of the pulmonary alveolus on the first day after injection, and the distribution of muscle, heart, brain and spleen was found seven days after injection. When the body tissue is damaged, bone marrow MSCs can reach the site of injury spontaneously through bone marrow mobilization, and differentiate into specific tissue cells in local microenvironment to participate in self repair. There is no unified statement on the mechanism of mobilizing bone marrow mesenchymal stem cells in the circulation to specific tissues or organs. The interaction of species and signal pathways.
In this study, the lentivirus particles expressed by the prepared double reporter gene system were labeled with MSCs in vitro. After detection of infected MSCs, the expression of RFP and Fluc reporter genes were stable, and the morphology was homogeneous and fibroid growth. The general biological characteristics of the cells were confirmed to be unaffected by the virus by the phenotype and differentiation of the cells in vitro. At the same time, combined with flow cytometry, the expression rate of RFP/Fluc double standard was close to 100%, which was a large number of cultured cells in the later period, and the living body imaging technique was used to monitor the distribution of the stable infected cells in the deep tissues of the mice.
【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】：2011
【分類號(hào)】：R329
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