CD28和4-1BB質(zhì)粒的構(gòu)建及在K562細(xì)胞上的表達(dá)
發(fā)布時(shí)間：2018-08-07 07:37
【摘要】：背景 鼻咽癌是東南亞地區(qū),尤其是我國(guó)南方各省最常見的惡性腫瘤之一,占廣東省頭頸部腫瘤的首位和全身惡性腫瘤死亡率的第3位。目前放射治療為鼻咽癌的首選治療手段,放化綜合治療5年生存率達(dá)50%—60%,但即使經(jīng)過(guò)正規(guī)根治的放化綜合治療,中晚期鼻咽癌患者的局部復(fù)發(fā)和遠(yuǎn)處轉(zhuǎn)移仍然是患者死亡的主要原因,并且再程放化療的效果不理想,而毒副反應(yīng)卻明顯增加,病人生活質(zhì)量下降。因此,有必要為中晚期鼻咽癌患者提供一種安全有效的二線治療方法。腫瘤生物治療隨著現(xiàn)代分子生物學(xué)技術(shù)和腫瘤免疫學(xué)的飛速發(fā)展不斷得到充實(shí)與更新,目前已成為繼手術(shù)、化療、放療之后的第四大治療手段,而被廣大醫(yī)務(wù)工作者和患者接受。過(guò)繼性細(xì)胞免疫治療是腫瘤免疫治療的一個(gè)重要分支,它是通過(guò)輸注抗腫瘤免疫效應(yīng)細(xì)胞的方法增強(qiáng)腫瘤患者的免疫功能達(dá)到抗腫瘤的目的,過(guò)繼性細(xì)胞免疫治療分為:非特異性免疫治療(腫瘤患者自體外周血CIK細(xì)胞治療技術(shù))已廣泛用于臨床,有效率在30%左右,經(jīng)過(guò)CIK細(xì)胞治療技術(shù)改良后有效率還可望提高;特異性免疫治療,是對(duì)某種腫瘤細(xì)胞有針對(duì)殺傷作用的免疫治療方法,療效優(yōu)于非特異性免疫治療,具有廣闊的臨床應(yīng)用前景。 EB病毒是一種與多種腫瘤發(fā)生發(fā)展密切相關(guān)的外部致病因子,它在多種腫瘤細(xì)胞中存在并且與這些腫瘤的發(fā)生發(fā)展密切相關(guān),已經(jīng)證實(shí),鼻咽癌與EB病毒的感染密切相關(guān),該病毒的存在為以細(xì)胞毒性T細(xì)胞(CTL)為基礎(chǔ)的免疫治療提供了一個(gè)潛在靶位,目前許多關(guān)于EB病毒相關(guān)腫瘤免疫治療的研究都是針對(duì)其表達(dá)的抗原LMP2的。但這種特異性的免疫治療方法,需要在體外培養(yǎng)出大量和高效的特異殺傷性CTL,經(jīng)過(guò)處理后再輸入人體進(jìn)行治療,傳統(tǒng)的PBMC/OKT-3法培養(yǎng)擴(kuò)增出的EBV特異性CTL存在特異性抗腫瘤免疫反應(yīng)低下的問(wèn)題,在CTL數(shù)量、活性和穩(wěn)定性方面均有缺陷,其中一個(gè)重要原因是T細(xì)胞共刺激分子表達(dá)不足或缺陷。CD28和4-1BB是迄今發(fā)現(xiàn)的T細(xì)胞上兩種最重要的共刺激分子,前者表達(dá)于靜止T細(xì)胞,優(yōu)先誘導(dǎo)CD4+T細(xì)胞活性;后者表達(dá)于活化T細(xì)胞,優(yōu)先誘導(dǎo)CD8+T細(xì)胞活性。它們分別與抗原遞呈細(xì)胞(APC)上的B7(CD80)和4-1BBL結(jié)合,協(xié)同MHC-Ag信號(hào),提高腫瘤細(xì)胞免疫原性,促進(jìn)T細(xì)胞活化、增殖、分化和凋亡,在T細(xì)胞的持續(xù)活化過(guò)程中發(fā)揮重要作用。 這種共刺激分子介導(dǎo)的協(xié)同刺激通路雖然是非特異性的,但它們?cè)诮閷?dǎo)免疫反應(yīng)的過(guò)程中相互調(diào)節(jié),發(fā)揮互補(bǔ)作用,對(duì)于T細(xì)胞的激活是不可缺少的。因此尋找一種能穩(wěn)定表達(dá)這兩種共刺激分子的實(shí)驗(yàn)方法,是研究其作為CTL體外擴(kuò)增系統(tǒng)的基礎(chǔ),也為鼻咽癌及其它腫瘤的免疫治療提供了條件。 實(shí)驗(yàn)?zāi)康?構(gòu)建和克隆CD28和4-1BB分子的重組質(zhì)粒,并在白血病細(xì)胞(K562)上穩(wěn)定表達(dá)。 方法 1、培養(yǎng)人白血病細(xì)胞(K562)。 2、合成人CD28分子的基因序列,亞克隆于pcDNA3.1-Neo構(gòu)建重組表達(dá)載體,并將重組質(zhì)粒轉(zhuǎn)化大腸桿菌(E.coli DH-5α菌株)。 3、合成人4-1BB分子的基因序列,亞克隆于pcDNA3.1-Neo構(gòu)建重組表達(dá)載體,并將重組質(zhì)粒轉(zhuǎn)化大腸桿菌(E.coli DH-5α菌株)。 4、于大腸桿菌內(nèi)大量擴(kuò)增和提取CD28和4-1BB重組質(zhì)粒。 5、構(gòu)建質(zhì)粒轉(zhuǎn)染的K562細(xì)胞。 6、篩選轉(zhuǎn)染的K562細(xì)胞。 7、用流式細(xì)胞儀檢測(cè)CD28和4-1BB在K562細(xì)胞上的表達(dá)。 結(jié)果 1、合成人CD28分子的基因序列,經(jīng)DNA序列分析證實(shí)合成的基因序列為人CD28編碼序列。 2、將合成產(chǎn)物與pcDNA3.1-Neo載體相連接得到重組質(zhì)粒,經(jīng)酶切電泳結(jié)果證實(shí)含有目的基因片段(即CD28基因序列)。 3、合成人4-1BB分子的基因序列,經(jīng)DNA序列分析證實(shí)合成的基因序列為人4-1BB編碼序列。 4、將合成產(chǎn)物與pcDNA3.1-Neo載體相連接得到重組質(zhì)粒,經(jīng)酶切電泳結(jié)果證實(shí)含有目的基因片段(即4-1BB基因序列)。 5、采用流式細(xì)胞儀檢測(cè)顯示有29.54%轉(zhuǎn)染的K562細(xì)胞表達(dá)共刺激分子CD28。 6、采用流式細(xì)胞儀檢測(cè)顯示有28.31%轉(zhuǎn)染的K562細(xì)胞表達(dá)共刺激分子4-1BB。 結(jié)論本研究表明運(yùn)用此種實(shí)驗(yàn)方法可成功地克隆并在白血病細(xì)胞K562上穩(wěn)定表達(dá)共刺激分子CD28和4-1BB,并以此作為CTL體外擴(kuò)增系統(tǒng)的實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:background
Nasopharyngeal carcinoma is one of the most common malignant tumors in Southeast Asia, especially in southern provinces of our country. It accounts for third of the first and third malignant tumors of the head and neck cancer in the south of China. Radiation therapy is the first choice for nasopharyngeal carcinoma. The 5 year survival rate of radiotherapy is 50% - 60%, but even after a regular radical cure Combined treatment, local recurrence and distant metastasis of patients with advanced nasopharyngeal carcinoma are still the main causes of death, and the effect of retherapy and chemotherapy is not ideal, but the side effects are obviously increased and the quality of life of the patients is decreased. Therefore, it is necessary to provide a safe and effective second line treatment for patients with middle and late nasopharyngeal carcinoma. With the rapid development of modern molecular biology technology and tumor immunology, treatment has been continuously enriched and updated. It has become the fourth major therapeutic means following surgery, chemotherapy and radiotherapy, and is accepted by the vast majority of medical workers and patients. Adoptive cellular immunotherapy is an important branch of the tumor immunotherapy. The method of anti tumor immune response cells enhances the immune function of the tumor patients to achieve the purpose of anti-tumor. Adoptive cell immunotherapy is divided into: non specific immunotherapy (tumor patients autologous peripheral blood CIK cell therapy) has been widely used in clinical, effective in 30% left right, after the improvement of CIK cell therapy efficiency is also effective. It is expected to be improved; specific immunotherapy is a method of immunotherapy against some tumor cells, which is better than non specific immunotherapy, and has a broad prospect of clinical application.
EB virus is an external pathogenic factor closely related to the development of a variety of tumors. It exists in a variety of tumor cells and is closely related to the development of these tumors. It has been proved that nasopharyngeal carcinoma is closely related to the infection of EB virus. The presence of this virus provides an immunotherapy based on cytotoxic T cells (CTL). At present, many of the potential targets of EB virus related tumor immunotherapy are aimed at its expressed antigen LMP2. However, this specific immunotherapy needs to develop a large and efficient specific killer CTL in vitro, and then enter the human body for treatment after treatment and the traditional PBMC/OKT-3 method to develop the amplified EB. V specific CTL has a specific anti tumor immune response problem, which has defects in the number, activity and stability of CTL. One of the important reasons is that the deficiency of T cell costimulatory molecules or defective.CD28 and 4-1BB are the two most important co stimulators on the T cells found so far. The former is expressed in static T cells and is preferred to lure them. The activity of CD4+T cells is guided; the latter is expressed in the activation of T cells and gives priority to inducing the activity of CD8+T cells. They are combined with B7 (CD80) and 4-1BBL on the antigen presenting cell (APC), together with the MHC-Ag signal, to enhance the immunogenicity of the tumor cells, promote the activation, proliferation, differentiation and apoptosis of the T cells, and play an important role in the continuous activation of T cells.
The co stimulatory pathway mediated by these co stimulators is nonspecific, but they are complementary to each other during the mediated immune response and play a complementary role in the activation of T cells. Therefore, an experimental method for the stable expression of these two co stimulators is studied as an in vitro CTL amplification system. The foundation of the system also provides conditions for immunotherapy of nasopharyngeal carcinoma and other tumors.
Experimental purpose
We constructed and cloned CD28 and 4-1BB recombinant plasmids and expressed them on leukemia cells (K562).
Method
1, human leukemia cells (K562) were cultured.
2. The gene sequence of human CD28 was synthesized and subcloned into pcDNA3.1-Neo to construct the recombinant expression vector. The recombinant plasmid was transformed into E.coli DH-5a strain.
3. The gene sequence of human 4-1BB molecule was synthesized and subcloned into pcDNA3.1-Neo to construct the recombinant expression vector. The recombinant plasmid was transformed into E.coli DH-5a strain.
4, a large number of CD28 and 4-1BB recombinant plasmids were amplified and extracted in E. coli.
5, the plasmid transfected K562 cells were constructed.
6, the transfected K562 cells were screened.
7, the expression of CD28 and 4-1BB on K562 cells was detected by flow cytometry.
Result
1, the sequence of synthetic human CD28 gene is confirmed by DNA sequence analysis, and the synthesized gene sequence is human CD28 coding sequence.
2. The recombinant plasmid was linked with pcDNA3.1-Neo vector, and the result of enzyme digestion electrophoresis confirmed that the recombinant plasmid contained the target gene fragment (CD28 gene sequence).
3, the sequence of synthetic human 4-1BB gene is confirmed by DNA sequence analysis, and the synthesized gene sequence is human 4-1BB coding sequence.
4. The recombinant plasmid was linked with pcDNA3.1-Neo vector, and the recombinant plasmid was confirmed to contain the target gene fragment (i.e. 4-1BB gene sequence) by enzyme digestion electrophoresis.
5, flow cytometry showed that 29.54% of the transfected K562 cells expressed costimulatory molecule CD28..
6, flow cytometry showed that 28.31% of the transfected K562 cells expressed costimulatory molecule 4-1BB..
Conclusion this study shows that this method can be successfully cloned and stable expression of costimulatory molecules CD28 and 4-1BB on leukemic cell K562, which can be used as an experimental basis for the CTL amplification system.
【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R739.63
[Abstract]:background
Nasopharyngeal carcinoma is one of the most common malignant tumors in Southeast Asia, especially in southern provinces of our country. It accounts for third of the first and third malignant tumors of the head and neck cancer in the south of China. Radiation therapy is the first choice for nasopharyngeal carcinoma. The 5 year survival rate of radiotherapy is 50% - 60%, but even after a regular radical cure Combined treatment, local recurrence and distant metastasis of patients with advanced nasopharyngeal carcinoma are still the main causes of death, and the effect of retherapy and chemotherapy is not ideal, but the side effects are obviously increased and the quality of life of the patients is decreased. Therefore, it is necessary to provide a safe and effective second line treatment for patients with middle and late nasopharyngeal carcinoma. With the rapid development of modern molecular biology technology and tumor immunology, treatment has been continuously enriched and updated. It has become the fourth major therapeutic means following surgery, chemotherapy and radiotherapy, and is accepted by the vast majority of medical workers and patients. Adoptive cellular immunotherapy is an important branch of the tumor immunotherapy. The method of anti tumor immune response cells enhances the immune function of the tumor patients to achieve the purpose of anti-tumor. Adoptive cell immunotherapy is divided into: non specific immunotherapy (tumor patients autologous peripheral blood CIK cell therapy) has been widely used in clinical, effective in 30% left right, after the improvement of CIK cell therapy efficiency is also effective. It is expected to be improved; specific immunotherapy is a method of immunotherapy against some tumor cells, which is better than non specific immunotherapy, and has a broad prospect of clinical application.
EB virus is an external pathogenic factor closely related to the development of a variety of tumors. It exists in a variety of tumor cells and is closely related to the development of these tumors. It has been proved that nasopharyngeal carcinoma is closely related to the infection of EB virus. The presence of this virus provides an immunotherapy based on cytotoxic T cells (CTL). At present, many of the potential targets of EB virus related tumor immunotherapy are aimed at its expressed antigen LMP2. However, this specific immunotherapy needs to develop a large and efficient specific killer CTL in vitro, and then enter the human body for treatment after treatment and the traditional PBMC/OKT-3 method to develop the amplified EB. V specific CTL has a specific anti tumor immune response problem, which has defects in the number, activity and stability of CTL. One of the important reasons is that the deficiency of T cell costimulatory molecules or defective.CD28 and 4-1BB are the two most important co stimulators on the T cells found so far. The former is expressed in static T cells and is preferred to lure them. The activity of CD4+T cells is guided; the latter is expressed in the activation of T cells and gives priority to inducing the activity of CD8+T cells. They are combined with B7 (CD80) and 4-1BBL on the antigen presenting cell (APC), together with the MHC-Ag signal, to enhance the immunogenicity of the tumor cells, promote the activation, proliferation, differentiation and apoptosis of the T cells, and play an important role in the continuous activation of T cells.
The co stimulatory pathway mediated by these co stimulators is nonspecific, but they are complementary to each other during the mediated immune response and play a complementary role in the activation of T cells. Therefore, an experimental method for the stable expression of these two co stimulators is studied as an in vitro CTL amplification system. The foundation of the system also provides conditions for immunotherapy of nasopharyngeal carcinoma and other tumors.
Experimental purpose
We constructed and cloned CD28 and 4-1BB recombinant plasmids and expressed them on leukemia cells (K562).
Method
1, human leukemia cells (K562) were cultured.
2. The gene sequence of human CD28 was synthesized and subcloned into pcDNA3.1-Neo to construct the recombinant expression vector. The recombinant plasmid was transformed into E.coli DH-5a strain.
3. The gene sequence of human 4-1BB molecule was synthesized and subcloned into pcDNA3.1-Neo to construct the recombinant expression vector. The recombinant plasmid was transformed into E.coli DH-5a strain.
4, a large number of CD28 and 4-1BB recombinant plasmids were amplified and extracted in E. coli.
5, the plasmid transfected K562 cells were constructed.
6, the transfected K562 cells were screened.
7, the expression of CD28 and 4-1BB on K562 cells was detected by flow cytometry.
Result
1, the sequence of synthetic human CD28 gene is confirmed by DNA sequence analysis, and the synthesized gene sequence is human CD28 coding sequence.
2. The recombinant plasmid was linked with pcDNA3.1-Neo vector, and the result of enzyme digestion electrophoresis confirmed that the recombinant plasmid contained the target gene fragment (CD28 gene sequence).
3, the sequence of synthetic human 4-1BB gene is confirmed by DNA sequence analysis, and the synthesized gene sequence is human 4-1BB coding sequence.
4. The recombinant plasmid was linked with pcDNA3.1-Neo vector, and the recombinant plasmid was confirmed to contain the target gene fragment (i.e. 4-1BB gene sequence) by enzyme digestion electrophoresis.
5, flow cytometry showed that 29.54% of the transfected K562 cells expressed costimulatory molecule CD28..
6, flow cytometry showed that 28.31% of the transfected K562 cells expressed costimulatory molecule 4-1BB..
Conclusion this study shows that this method can be successfully cloned and stable expression of costimulatory molecules CD28 and 4-1BB on leukemic cell K562, which can be used as an experimental basis for the CTL amplification system.
【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R739.63
【共引文獻(xiàn)】
相關(guān)期刊論文 前9條
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