Screening Of Differentially Expressed Genes Of PBMC In Ovarian Cancer Patients And Construction Of Single-chain Variable Fragment Antibody Library Of Ovarian Cancer

Background: Ovarian cancer(OC)is one of the most common malignant tumors in women.Although the morbidity rate of OC is lower than that of cervical cancer and uterine cancer,it's the most lethal gynecologic malignancy in female.The early symptoms of ovarian cancer always vague and as a result of the lack of reliable diagnosis methods in the early stage,almost 70% of patients are diagnosed at an advanced stage.The early diagnosis of ovarian cancer is the most vital factor related to the prognosis of the patients.Gene chip technology has become one of the methods for early diagnosis of ovarian cancer.Peripheral blood mononuclear cells(PBMC)are comprised of lymphocytes and mononuclear cells,and also include some of circulating tumor cells(CTCs)and tumor stem cells(TSCs).Because of this,the changes of microenvironment in tumor patients can be reflected in PBMC.In this research,PBMC from the peripheral blood were divided into two groups,ovarian cancer samples and matched controls.Gene expression profilings were detected by using gene chip technology.By the detection of Gene expression profilings,not only contributes to the early diagnosis of ovarian cancer,and also reveals the mechanism of occurrence and metastasis of ovarian cancer to some certain extent.Single-chain fragment variable(sc Fv)is a combination of heavy-chain variable region and light-chain variable region with a linker of 15-20 amino acid fragments.Single-chain fragment variable is the minimal form of functional antibodies.It has characteristics such as lower molecular weight,strong tissue penetration capability,and the weaker immunogenicity and is widely used as diagnostic tools and therapeutic agents of tumor.For instance,sc Fv labeled with radionuclide have the extensive application for in situ imaging of tumor tissue,or sc Fv connected with cytotoxic or chemotherapeutic drugs for cancer therapy.In this research,we successful constructed a single-chain variable fragment antibody library of ovarian cancer patients by using the yeast surface display technology,and screened the library by using ovarian cancer cell line.Providing the fundamental of acquisition of sc Fv,and this perhaps has benefit use to clinical diagnoses and therapy.Purpose: Chapter 1: The gene expression profiles of peripheral blood mononuclear cells of ovarian cancer patients and normal controls were detected by using gene chip technology,and the differentially expressed genes of PBMC of patients were screened.Gene Ontology analysis and gene pathway analysis were performed to the differentially expressed genes.Chapter 2: To construct a sc Fv antibody library for ovarian cancer patiens by using yeast surface display technology and to evaluate the libaray capacity.Screening the library displayed on yeast cell surface by using ovarian cancer cell line A2780.To establish the fundamental to achieve the sc Fv antibodies for ovarian cancer cells with high affinity.Method: Chapter 1: We extracted the peripheral blood mononuclear cells from 8patients who were diagnosed as ovarian cancer and 4 healthy females,and extracted the total RNA from the PBMCs.For the purpose to eliminate the differences between samples,we pairwise mixed the samples in patients group,and finally obtained 4samples in patients group.We used commercial Affymetrix? Human Genome U219 Array Strip to discover the expression of transcripts in two groups,and used online softwares to analysis the DEGs.Chapter 2: We applied the peripheral blood mononuclear cells from 13 patients who were diagnosed as ovarian cancer,and extracted the total RNA of the PBMCs by TRIzol reagent.We amplified c DNA gene library using reverse transcriptase PCR,and amplified light chain variable region and of heavy chain variable region gene fragment using previous designed primers.The VH gene fragment and VL gene fragment were connected by SOE-PCR.In this paper,we exploited the potential of the in vivo homologous gap-repair apparatus of yeast,and co-transformed VH-VL fragment and linearized vector p YD1 into the yeast.We screened the library with a tryptophan free selective culture-medium after co-transform.In the induced medium with galactose,the yeast expressed single-chain antibody on the surface of yeast cells,which means established the antibody library.Then,the ovarian cancer cell line A2780 was used in combination with the yeast cells expressing sc Fv,and the yeast strains combined with A2780 cells were screened by flow cytometry.Result : Chapter 1: After analysed the microarray results,we identified 1158 differentially expressed genes(Fold Change>2,p<0.05)in the PBMC of the patients with ovarian cancer compared with the PBMC of the healthy.Among the differentially expressed genes,311 genes were upregulated and 847 genes were downregulated.The most obvious 5 genes in upregulated group were THBS1,NR4A2,BTG1,ADM and MIR22,and the most obvious 5 genes in downregulated group were GIMAP8,CX3CR1,GIMAP4,ST8SIA4 and MYOM2.Pathway analysis of differentially expressed genes revealed that the pathways involved were mainly distributed in the signal transduction system and the immune system.In the GO analysis of differentially expressed genes,we had several conclusions.Firstly,cellular component analysis was mainly involving cellular components and cell organelles.Secondly,molecular function analysis was mainly involving binding and catalytic activity.Thirdly,biological process analysis was mainly involving cellular process and metabolic process.After functional categories analysis of differentially expressed genes,6 genes were screened out with significant differences in the intersection of the apoptotic categories and the immune system process categories.Chapter 2: We successfully amplified VH gene fragment and VL gene fragment by PCR,and co-transformed the conneted VH-VL fragment and linearized vector p YD1 into the yeast.In the selective culture-medium,the growth of yeast colony was observed.The size of the constructed sc Fv antibody library is,4×109 of the ? light chain library and8×109 of the ? light chain library.By using the ovarian cancer cell line A2780 to screen the antibody library,we obtained that,21 strains of the ? light chain library and 89 strains of the ? light chain library.Conclusion: Chapter 1: There were significant differences in the gene expression profiles of PBMC in patients with ovarian cancer and healthy controls.Chapter 2: We successfully constructed sc Fv antibody library of ovarian cancer by using the yeast surface display technology,expressed single chain antibodies on the surface of yeast in induced medium,and preliminary screened the library by using ovarian cancer cell line A2780.To provide a foundation for further achieve high affinity single chain antibodies to ovarian cancer.