CD28和4-1BB质粒的构建及在K562细胞上的表达
发布时间：2018-08-07 07:37
【摘要】：背景 鼻咽癌是东南亚地区,尤其是我国南方各省最常见的恶性肿瘤之一,占广东省头颈部肿瘤的首位和全身恶性肿瘤死亡率的第3位。目前放射治疗为鼻咽癌的首选治疗手段,放化综合治疗5年生存率达50%—60%,但即使经过正规根治的放化综合治疗,中晚期鼻咽癌患者的局部复发和远处转移仍然是患者死亡的主要原因,并且再程放化疗的效果不理想,而毒副反应却明显增加,病人生活质量下降。因此,有必要为中晚期鼻咽癌患者提供一种安全有效的二线治疗方法。肿瘤生物治疗随着现代分子生物学技术和肿瘤免疫学的飞速发展不断得到充实与更新,目前已成为继手术、化疗、放疗之后的第四大治疗手段,而被广大医务工作者和患者接受。过继性细胞免疫治疗是肿瘤免疫治疗的一个重要分支,它是通过输注抗肿瘤免疫效应细胞的方法增强肿瘤患者的免疫功能达到抗肿瘤的目的,过继性细胞免疫治疗分为:非特异性免疫治疗(肿瘤患者自体外周血CIK细胞治疗技术)已广泛用于临床,有效率在30%左右,经过CIK细胞治疗技术改良后有效率还可望提高;特异性免疫治疗,是对某种肿瘤细胞有针对杀伤作用的免疫治疗方法,疗效优于非特异性免疫治疗,具有广阔的临床应用前景。 EB病毒是一种与多种肿瘤发生发展密切相关的外部致病因子,它在多种肿瘤细胞中存在并且与这些肿瘤的发生发展密切相关,已经证实,鼻咽癌与EB病毒的感染密切相关,该病毒的存在为以细胞毒性T细胞(CTL)为基础的免疫治疗提供了一个潜在靶位,目前许多关于EB病毒相关肿瘤免疫治疗的研究都是针对其表达的抗原LMP2的。但这种特异性的免疫治疗方法,需要在体外培养出大量和高效的特异杀伤性CTL,经过处理后再输入人体进行治疗,传统的PBMC/OKT-3法培养扩增出的EBV特异性CTL存在特异性抗肿瘤免疫反应低下的问题,在CTL数量、活性和稳定性方面均有缺陷,其中一个重要原因是T细胞共刺激分子表达不足或缺陷。CD28和4-1BB是迄今发现的T细胞上两种最重要的共刺激分子,前者表达于静止T细胞,优先诱导CD4+T细胞活性;后者表达于活化T细胞,优先诱导CD8+T细胞活性。它们分别与抗原递呈细胞(APC)上的B7(CD80)和4-1BBL结合,协同MHC-Ag信号,提高肿瘤细胞免疫原性,促进T细胞活化、增殖、分化和凋亡,在T细胞的持续活化过程中发挥重要作用。 这种共刺激分子介导的协同刺激通路虽然是非特异性的,但它们在介导免疫反应的过程中相互调节,发挥互补作用,对于T细胞的激活是不可缺少的。因此寻找一种能稳定表达这两种共刺激分子的实验方法,是研究其作为CTL体外扩增系统的基础,也为鼻咽癌及其它肿瘤的免疫治疗提供了条件。 实验目的 构建和克隆CD28和4-1BB分子的重组质粒,并在白血病细胞(K562)上稳定表达。 方法 1、培养人白血病细胞(K562)。 2、合成人CD28分子的基因序列,亚克隆于pcDNA3.1-Neo构建重组表达载体,并将重组质粒转化大肠杆菌(E.coli DH-5α菌株)。 3、合成人4-1BB分子的基因序列,亚克隆于pcDNA3.1-Neo构建重组表达载体,并将重组质粒转化大肠杆菌(E.coli DH-5α菌株)。 4、于大肠杆菌内大量扩增和提取CD28和4-1BB重组质粒。 5、构建质粒转染的K562细胞。 6、筛选转染的K562细胞。 7、用流式细胞仪检测CD28和4-1BB在K562细胞上的表达。 结果 1、合成人CD28分子的基因序列,经DNA序列分析证实合成的基因序列为人CD28编码序列。 2、将合成产物与pcDNA3.1-Neo载体相连接得到重组质粒,经酶切电泳结果证实含有目的基因片段(即CD28基因序列)。 3、合成人4-1BB分子的基因序列,经DNA序列分析证实合成的基因序列为人4-1BB编码序列。 4、将合成产物与pcDNA3.1-Neo载体相连接得到重组质粒,经酶切电泳结果证实含有目的基因片段(即4-1BB基因序列)。 5、采用流式细胞仪检测显示有29.54%转染的K562细胞表达共刺激分子CD28。 6、采用流式细胞仪检测显示有28.31%转染的K562细胞表达共刺激分子4-1BB。 结论本研究表明运用此种实验方法可成功地克隆并在白血病细胞K562上稳定表达共刺激分子CD28和4-1BB,并以此作为CTL体外扩增系统的实验基础。
[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.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：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.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.63
【共引文献】
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1 马肖容,张王刚,杨惠云,田玮;急性白血病外周血T细胞CD28和CD137的表达[J];第四军医大学学报;2003年19期
2 周文;张鹏;宋世辉;温昱鹏;吕扬;张健;;300例肺癌患者淋巴细胞计数结果分析[J];中国肺癌杂志;2009年10期
3 吴宜嘉;;流式细胞仪在淋巴瘤患者外周血T细胞亚群诊断中的应用研究[J];海南医学院学报;2010年12期
4 吉春宇,杨异,赵珩,陈文虎;手术对肺癌患者外周血T细胞亚群的影响[J];上海医学;2004年10期
5 张仪;韩波;;CD_(28)在疾病治疗中的作用研究进展[J];实用儿科临床杂志;2008年15期
6 王群,张利宁,孙汶生,丁培芳,刘军莉,张，
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