Experimental Study Of SMMC-7721 Human HCC Cells With Kupffer Cells Transduced With An Recombinant Adenoviral Vector Expressing Interleukin 12 In Vitro And In Vivo

Objectives:(1)To isolate and purify Kupffer Cell(KC)from Sprague-Dawley(SD) rat liver and receive highly purified and active KC.(2)To explore apoptosis-inducing effect of Kupffer Cells transduced with an recombinant Adenoviral Vector expressing Interleukin 12 on SMMC-7721 human HCC cells.(3)In nude mice bearing SMMC-7721 tumors,to study the growth-suppression of HCC tumors with the effect of Kupffer Cells transduced with an recombinant Adenoviral Vector expressing Interleukin 12;in addition, to observe immune system on SMMC-7721 bearing tumor mice.Methods:(1)A combined "collagenase IV & pronase E" perfusion,in situ and ex vivo, and then the discontinuous density gradient centrifugation using Nycodenz was performed to obtain the Nonparenchymal cells(NPC)from SD rat liver.Further,the method of selective adherence was used to purify and recover the KC from NPC.The activity and quantity of cells was judged by Trypan blue exclusion test.To identify the KC,we observed the phenomenon of auto-fluoresence,KC's phagocytosis action and performed immunocytochemistry with the mouse anti-rat monoclonal antibody ED-2.(2) To complete the Adv-mIL-12 and Adv-EGFP replicated and expressed in QBI-293A cells and to detect these potency.To detect quantitatively the level of IL-12 about Kupffer Cells transduced with Adv-mIL-12 and Adv-EGFP.Different formal KC and SMMC-7721 cells were seeded in 96-well plates at ratioes 20:1,10:1,5:1,2.5: 1 and were divided into 7 groups:SMMC-7721,KC,KC-Adv-EGFP,KC-Adv-mIL-12,KC+SMMC-7721,KC-Adv-EGFP+SMMC-7721 and KC-Adv-mIL-12+ SMMC-7721.The cells viability were measured by MTT assay using the spectrophotometrical absorbance at 570nm every day for a total of 7 days.Different formal KC and SMMC-7721 cells were seeded in 24-well plates at a ratio 5:1 and were divided into 4 groups:SMMC-7721,KC+SMMC-7721,KC-Adv-EGFP+ SMMC-7721,KC-Adv-mIL-12+SMMC-7721.The supernatants were harvested after 1,2,3,4,5,6,7days,then supematant samples were testified with VEGF ELISA kit to detect quantitatively the level of VEGF.The supernatants were harvested about different formal KC after 24,48,72hours,and let these supernatants affect SMMC-7721. Forty-eight hours after effection,cells were harvested and analyzed using by FCM.(3) Female BALB/C mice(5 each group)were inoculated subcutaneously on their right thighs with 2×10~6 SMMC-7721 cells.10 days later,when the tumors grew up to 5mm, the mice were intratumorally and extratumorally injected with Adv-mIL-12(1×10~8 pfu, every 4 days,for 3 times)and different formal KCs(2×10~6,every 4 days,for 3 times), 100μl/each tumor respectively.Tumor progression and regression was monitored daily. Tumor volumes(in cubic centimeters)were measured with a vernier caliper before every injection and 7 days after the last injection.7 days after the last injection,these animals were sacrificed,peripheral blood was taken out from their eye-frame vessels,spleens of all mice were broken out and weighed.The numbers of white blood cell,lymphocyte,monocytes,neutrophil were detected.Index of spleen(SI)=weight of spleen/body weight.The SMMC-7721 tumor were carefully removed and RT-PCR was used to test VEGFmRNA's level,immunocytochemistry was used to detect masculinal cells of PCNA,Bcl-2,CD34.To observe inhibition ratio and MVD about different tumor group.Results:(1)The yield of KC per rat were 1.0～1.5×10~7,in which over 93%viable by Trypan blue exclusion test and over 95%were positive for ED-2.(2)These potencies about Adv-mIL-12 and Adv-EGFP replicated in QBI-293A cells were 2.0×10~9pfu/ml and 3.0×l0~9pfu/ml.At 24h,uninfected KC and adv-EGFP-infected KC produced very low levels of IL-12 respectively,whereas a high level of secretion of IL-12(564.29±31.24 pg/ml),10-fold of which of controls,was detected in the adv-mIL-12-infected KC.The transfection efficiency about adv-EGFP-infected KC with the same potency was over 90%at 24h.The MTT assay showed that the inhibition ratio about SMMC-7721 was 50.12%in the experimental group(KC-Adv-mIL-12+SMMC-7721)at 4d,which showed conspicuous statistical significance compared which of control groups'(KC+SMMC-7721 25%).ELISA test demonstrated that the concentration of VEGF(the experimental group)was very low level compared with control groups'.The FCM showed that more apoptosis cells were detected in the the experimental group(KC-Adv-mIL-12 supematants+SMMC-7721)compared with control groups.The sub-G1 surge in the the experimental group was 12.2%.(3)The tumor volumes in the mice injected with KC-Adv-mIL-12 were significantly diminished than that with NS,Adv-mIL-12,KC,or KC-Adv-EGFP(KC-Adv-mIL-1261.84 mm~3;NS 637.75 mm~3). KC-Adv-mIL-12 enhanced significantly the indexes of spleens and the numbers of immune cells in peripheral blood compared with control groups.The HE dyeing about tumor tissue showed that the heteromorphism about tumour cell was diminished in the the experimental group compared with control groups.RT-PCR demonstrated that the expression of VEGFmRNA was knocked down significantly in the the experimental group,and the experimental group significantly down-regulated expression of PCNA,Bcl-2 in tumors infected with KC-Adv-mIL-12 compared with NS,Adv-mIL-12,KC,KC-Adv-EGFP,and there also down-regulated expression of CD34.Conclusions:(1)The activity as well as purity of KC,which be attained from SD rat by the methods of combined "collagenaseⅣ& pronase E" perfusion and discontinuous density gradient centrifugation using Nycodenz and selective adherence were satisfactory and these KC can be used in further study.(2)The adenoviral vector can transfer the IL-12 gene to KC in vitro effectually and the modified KC highly expressed IL-12 protein.KC-Adv-mIL-12 can inhibit the proliferation of SMMC-7721 human HCC cells and can increas the percentage of apoptotic cells.(3)KC-Adv-mIL-12 can inhibit tumor growth significantly and can enhance organismal immunologic system markedly.