The Monoclonal Antibody Against Human CD133 And CD133 Expression On Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related death worldwide, one of the most common malignant tumors in China as well. It is difficult to cure because of its high-grade malignancy and rapid progress.The theory of tumor stem cell (TSC), or cancer stem cell (CSC) is well established based on numerous data that accumulated in dozens of decades.TSC is a small population within tumors, and belongs to adult stem cells with characteristic of self-renewing, differentiation and malignant proliferation. More and more reports demonstrated that CD133+ cell subpopulation from tumor was identified as tumour initiating cells. In a way, the CD133+ cancer stem cells are the origin of chemoresistance, apoptosis resistance, metastasis and recurrence of various kinds of cancers. TSC was found in many types of tumors, including leukemia, breast cancer, brain cancer, colorectal cancer, pancreatic cancer. It is reported that TSC is involved in the pathologic process of hepatocarcinoma as well.Many kinds of biomarkers of TSC were found on different types of tumors.CD133 is one of the most important biomarkers to identify TSC.CD133 gene is located in chromosome 4. The full length CD133 gene is 160Kb, and it is composed of 27 exons with 1589bp of ORF(open reading frame), edcoding 5-transmembrane glycoprotein with 865 aminoacide residues, and its expected molecule weight is 120KD.Many reports demonstrated that CD133 is a perfect surface marker to identify TSC from many types of tumor. CD133+ cells are associated with tumor metastasis and tumor microvessels formation. Tumor relapse could be detected by quantitative CD133 expression in mRNA level. CD133 is also the surface biomarker of TSC of hepatocarcinoma. Hence, our study is focused on the expression of CD133 of hepatocacinoma and its clinical significance.First, we break through the bottle neck of long gene (>2Kb) cloning by using high quality fetal liver cDNA library as PCR template and cloning 3 overlaped fragments. And then, all the fragments were linked together to get the full length CD 133 gene by PCR.Then, the full lentgh CD 133 gene was subcloned into eukaryotic expression vector pIRES/EGFP. And the mouse fibroblast cell L929 was infected with the pIRES/EGFP/CD133 expression vector. CD 133 antigen expressed on the L929 cell surface and it is critical for preparation of monoclonal antibody against human CD 133.However, the counterpart of CD133 is unknown. As our knowledge, the specific monoclonal antibody may ligate and convey the signal to a cell surface receptor. But there is no report of functional monoclonal antibodies against CD133. In this study, Balb/c mice were immunized with CD133+ monocytic cell line U937. Hybridoma was obtained with B-lymphocyte hybridoma technology. Briefly, splenocytes from immunized mice and myeloma cells SP2/0 were fused and screened with CD133 transfectant L-CD133 and its negative control cells L-mock. And then, the monoclonal antibody against human CD133 (6B6) was purified and its characteristic was identified. Similar to commercial antibody AC141, mAb6B6 could recognize CD133 molecules on different type of cell lines by immunostaining and flow cytometric analysis. MAb 6B6 could detect CD133+ cancer cells in colorectal samples by immunohistochemical staining. And then, we stimulated colorectal cell line Caco-2 that expressed CD133 with mAb 6B6 in vitro. Interestingly, we found that the proliferation of Caco-2cells was obviously inhibited in presence of MAb 6B6.Furthermore, we analyzed CD133 expression on hepatocellular carcinoma samples by immunohistochemical staining and discussed its clinical significance. MAb 6B6 could detect CD133+ cells on hepatocellular carcinoma tissue. The ratios of CD133+ cells were less than 5% and were lower than 1% on most of the samples. It is demonstrated that CD133+ hepatocellular carcinoma TSC is exist but it is a small population. Our data showed that the expression of CD133 is correlated with serum AFP level and pathological differentiation of hepatocellular carcinoma patients.(P<0.05) The higher lever of serum AFP and more poorly differentiation of hepatocellular carcinoma, the higher ratio of CD133+ cells. And there is no correlation of age, sex and tumor volumn with CD133+ TSC ratio.In conclusion, we have obtained a specific anti-human CD133 MAb with the potential for research and clinical applications in tumor diagnosis and immunotherapy.