Experimental Study Of Intralipid Effects On Transgene Expression With An Recombinant Adenoviral Vector Expressing Interleukin 12 In Vivo

Objective:(1)To complete the Adv-mIL-12 and Adv-EGFP replicated and expressed in QBI-293A cells and detect these potencies after purifying them.(2)To explore their relationship between the dose of Adv-mIL-12 and IL-12 gene expression after intraportal injection of different doses of Adv-mIL-12.(3)To explore the effects on IL-12 gene expression and toxicity of the body with prior application of Intralipid by intraportal injection of Adv-mIL-12.Methods:(1)QBI-293A cells were abundantly propagated in DMEM 10%,then we completed the Adv-mIL-12 and Adv-EGFP replicated and expressed in QBI-293A cells and utilized CsCl density gradient centrifugation and the TCID₅₀ method to purify adenoviruses and detect virus titer.(2)By intraportal injection of different doses of Adv-mIL-12(5×10⁸Pfu/Rat,1×10⁹Pfu/Rat 3×10⁹Pfu/Rat,5×10⁹Pfu/Rat,1×10¹⁰Pfu/Rat) after 72 hours,we explored IL-12 gene expression in serum and liver tissue with ELISA assay.At the same time,after 72 hours by prior tail vein injection different doses of Intralipid(0mg,50mg,100mg,150mg,200mg )before intraportal injection of 5×10⁸Pfu/Rat dose of Adv-mIL-12,we observed serum IL-12 concentration.Finally,we injected 5×10⁹Pfu/Rat of the Adv-mIL-12 with preadministration of 150mg of Intralipid,then after 72 hours,we observed IL-12 concentration in the liver,spleen,lung,kidney,heart with tissue homogenate and ELISA assay.(3)We divided four groups: Adv-mIL-12,Intralipid(Intralipid+Adv-mIL-12),Adv-EGFP and Saline group(as control group),then we observed the serum IL-12,IFN-γconcentration trends and Serum Levels of ALT,AST over time at the Adv-mIL-12,Adv-EGFP dose of 5×10⁹Pfu/Rat and Intralipid dose of 150mg/Rat.After 1 day,we sacrificed 2 rats in each group,used CD68 antibody on rat Kupffer cells by immunohistochemical staining and counted the positive cells under the microscope;Finally,we observed acute inflammatory cytokines TNF-α,IL-6 concentration in serum after 6 hours with ELISA assay.Results: (1)These potencies after purification about Adv-mIL-12 and Adv-EGFP replicated in QBI-293A cells were about 1.4×10¹⁰pfu/ml and 2.0×10¹⁰pfu/ml, respectively. (2)After 72 hours with intraportal injection of low doses of Adv-mIL-12(5×10⁸-1×10⁹Pfu/rat),we found that the expression of IL-12 gene was low in serum and liver and there existed a plateau for expression of IL-12 gene,When the dose increased to 3×10⁹Pfu/rat, the expressions of IL-12 gene in serum and liver was significantly increased,at least 3-fold of which at lower doses of Adv-mIL-12,which showed conspicuous statistical significance compared with that at lower doses of Adv-mIL-12. And with Adv-mIL-12 dose increasing,the increase of the expression of IL-12 gene was pronounced;When Adv-mIL-12 dose was 5×10⁸Pfu/Rat,along with the preadministration dose of Intralipid increasing,the level of serum IL-12 was significantly elevated,when the dose of Intralipid increased to 150mg,IL-12 content of the serum was 3-fold increase compared with that without applying the Intralipid.But at 200mg dose of Intralipid,we observed that the individual followed by respiratory distress in rats,more pronounced decrease in activity and eating situation;After 72 hours with 150mg of Intralipid and 5×10⁹Pfu/Rat of the Adv-mIL-12,we found that expression of IL-12 gene in the liver was the highest in five organs, which showed conspicuous statistical significance compared with other organs,so it can form local high concentration of IL-12 in the liver.(3)We found that the concentration of serum IL-12 in the Intralipid group was significantly higher than those in other groups at different time points,and serum IL-12 level in each groups was the highest at 72 hours,then gradually reduced,In the first 18 days,Intralipid group was still detected in serum IL-12 expression;Compared with the other groups,the concentration of serum IFN-γwas significantly increased at each time point but except at 24 hours;Except Saline group,the positive numbers of Kupffer cells in the other three groups were reduced,which was statistically significant between them,but the reduction of the number in the Intralipid group was significantly less than that in the Adv-mIL-12 group and Adv-EGFP group;At 24 hours,the serum levels of ALT and AST in the other three groups were apparently higher than that in the Saline group,especially in the Intralipid group,At 72 hours,the serum levels of ALT and AST in all groups decreased significantly and in the first 18 days,the serum levels of ALT and AST in three adenovirus groups returned to normal;Compared with Saline group,the acute inflammatory cytokines TNF-α,IL-6 serum concentration in the other three groups was significantly elevated at 6 hours,The TNF-αconcentration in the Intralipid group was the lowest in three adenovirus groups,but IL-6 concentration was opposite.Conclusions: (1)It can abundantly amplify Adv-mlL-12,Adv-EGFP in QBI-293A cells and after purification of adenovirus,we can get a high titer of adenovirus.(2) There exists threshold effects for the expression of IL-12 by intraportal injection of various dose of Adv-mIL-12,that is,with exceeding a certain dose of Adv-mIL-12,the expression of IL-12 in serum and liver may be substantially higher than before.(3)Compared with direct application of Adv-mIL-12,the serum IL-12 concentration by prior injection of a certain amount of Intralipid can be significantly elevated and the content of IL-12 in the liver is still the highest.in five organs.(4)With direct application of Adv-mIL-12 and the prior application of Intralipid in rats,the liver function can cause temporary injury.Simultaneously,compared with direct application of adenovirus,the TNF-αconcentration of the acute inflammatory cytokines in serum with the prior application of Intralipid is lower ,but the IL-6 concentration is opposite.