| Backgroud and AimsRenal cell carcinoma (RCC) is a common malignant tumor in the urinary system, especially the recurrence and metastasis of postoperative is common to patients with advanced stage tumor, RCC is not sensitive to radiotherapy and chemotherapy,the anti-effect of immunotherapy is also insignificant.so the prognosis of patients is poor. Bioresearch of renal tumor cell indicated that the morbility of RCC was associated with a biallelic loss of anti-oncogene VHL. This tumor suppressor gene is located on chromosome 3p25-26 with a total size of 15 kb and can encode von Hipple-Lindau protein, pVHL.The pVHL is found to bind with elongin B-C and CUL-2,and constitute VHL-ElonginB/C-CUL2(VBC) complex.This complex is confirmed that belong to the system of E3-ubiquitin-proteasome and involved in the degradation of a broad variety of proteins.then realizing cytokine inhibition and inhibiting cell proliferation by regulating cell cycle. Previous research had shown that one of the major functions of pVHL is its role in the ubiquitination machinery.Apart from its role in the proteasomal degradation, the pVHL could also inhibit IGF-IR-mediated signaling pivotal for the growth and development of RCC.This inhibition is accomplished by the ability of the 104-123 amino acid region of the (3-domain of the VHL molecule. Theβ-domain of the VHL molecule is sufficient to block the interaction between the cytoplasmic domain of the IGF-IR and its downstream signaling molecule PKC 8, thereby blocking PKCδkinase activity,which may ultimately affect cell proliferation and induce apoptosis of renal carcinoma cells. We constructed the recombinant AAV mediated secretive expression of NT4-TAT-6×His-VHLβfusion peptide(rAAV/NT4-TAT-6×His-VHLβ) on the basis of this mechanism, and displayed effect of toxicity and induced apoptosis of cell through transfection renal tumor cell in vitro. Next, we need establish implanted tumor model of human RCC, to observe anti-tumor effect of rAAV/NT4-TAT-6×His-VHLβ.Chick embryo chorioallantoic membrane(CAM) is formed between days 4 and 5 by partial fusion of chorion and allantois. Histologically, the CAM contains three layers,i.e.,the ectoderm attached to the shell membrane, the mesoderm enriched in blood vessels and stromal components, and the endoderm facing the allantoic cavity. Between days 6 and 7 of incubation,the CAM and its blood vessels cover the entire surface of yolk sac, and capillary become extremely rich along with the embryo age increase. The CAM is natural immunocompromised host and can tolerant temperature changes, we can detect biology behavior of tumor cell that sensitive to temperature. So the CAM is common as a implanted tumor experimental models.The aims of this study include:(1)To establish human renal cell carcinoma model on chick embryo chorioallantoic membrane and to study its biological characteristics; (2)To study the anti-tumor effect of rAAV/NT4-TAT-6×His-VHLβon the CAM implanted tumors.Materials and methods:(1) Recovery and cultivation of the human RCC cell line RLC-310,to collect cell in logarithmic phage and centrifuge,then adjust cell concentration with PBS, the cell concentration respectively is 1.0×106/30ul,2.0×106/30ul,4.0×106/30ul, 8.0×106/30ul.In the super clean bench, Open a window that the diameter is two centimeter at the air chamber of the 9th day's chick embryo, we can see the air chamber membrane at the bottom, then inject 1～2 drop bacteria free water through poke a small hole in the center of air chamber membrane. when the air chamber membrane and CAM separated completely,we remove the air chamber membrane and the CAM become visible. At this time,RLC-310 cell were inoculated on the chick embryo CAM. Closed the window with transparent adhesive tape and continue incubation Then we observe the growth.angiogenesis and morphology of the implanted tumors.(2)Collect cell in logarithmic phage and adjust cell concentration to 4.0×106/30ul.then expose CAM through open a window on the ninth day of chick embryo and RLC-310 cell were inoculated on the CAM.Three days later, we choose chick embryo that emerging implanted tumors and divided into three groups:the group of rAAV/NT4-TAT-6xHis-VHL(3;the group of AAV;the group of PBS. To observe growth of implanted tumors,and to measure the size of tumors on fifth days after treatment. Then complete detachment tumors,paraffin embedding and cut into slices,we observe the effect of anti-tumor through HE dyed and detect the expression of NT4-TAT-6×His-VHLβthrough immunofluorescence.Results:(1) We find that the tumors formation rate rise following the number of inoculated cell increase and confirm it is appropriate when we inoculate 4.0×106/30ul. We can observe growth of the implanted tumors on third after cells were inoculated on the CAM. Blood vessels centralized according radial pattern. The tumor on CAM reach a summit of growth on seventh day.The organizational structure and morphological of implanted tumor is consistence with human renal tumor.(2) The result display that the growth of implanted tumors become slower and the volume of tumors decrease in the group of rAAV/NT4-TAT-6×His-VHLβthan other two groups (P<0.05).The volume of three groups respectively is 43.85±16.60mm3,74.20±25.74mm3,82.35±33.96mm3.We find through HE dyed that tumors nodules are scatter and isolate, suggesting the growth ability of tumors is inhibited after treatment. Using fluorescence microscope, we can find bright fluorescent in the group of rAAV/NT4-TAT-6×His-VHLβ. while we can't perceive the fluorescent in contrast groups.So we detect the expression of NT4-TAT-6×His-VHLβfusion peptide in control groups through immunofluorescence.Conclusions(1)Human RCC model on CAM is of easy duplication and could be used to study the biological characteristics, induce angiogenesis and transfer mechanism of RCC, This model could be used in the experiment research for the therapy of human renal cell carcinoma.(2) rAAV/NT4-TAT-6×His-VHLp have anti-tumor effect to human RCC implanted tumors suggesting that it may be a novel tool for gene therapy of human renal cell carcinoma. |
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