Establishment Overall Amplification Technique Of Anti-hepatocellular Carcinoma Immune RNA And Study On Its Anti-hepatocellular Carcinoma Effect

The project team innovatively developed the first generation of anti-tumor immune ribonucleic acid(AT-iRNA)in the 1990 s,which is the total RNA extracted from immune organs after immunizing animals with tumor antigens.The first-generation AT-iRNA can stimulate the humoral and cellular immunity of cancer patients.Long-term and large-scale clinical application has proved that it can control metastasis and remove residual cancer cells in patients with primary lesions after surgical resection,and has good curative effect on many malignant tumors.But in the past decade,the production and sales of AT-iRNA have declined sharply.The main reason is that the nature of iRNA is unstable and easy to be degraded by RNA enzymes;the resulting immunity is not strong enough,specificity is not high enough,the application effect is not good,and the price is relatively expensive.The bottlenecks of the first generation AT-iRNA production technology include:(1)the preparation of tumor antigens is extremely rough,resulting in a significant reduction in the purity of tumor antigens;(2)the extraction process of AT-iRNA is simple,and there is no protective active iRNA program,resulting in a large number of iRNA degradation;(3)the content of active RNA is very small(3-5%),which seriously restricts the pharmacodynamics of anti-tumor immunotherapy;(4)the pharmacodynamics of the first generation AT-iRNA.The mechanism is not clear.In order to solve the above problems,this research is the first to develop a new generation of anti-tumor immune ribonucleic acid patent preparation process by integrating the innovation of molecular technology at home and abroad.Aiming at the problem that the content of active RNA in AT-iRNA is relatively low,which affects the therapeutic effect,this study established a technology for large-scale amplification of AT-iRNA by referring to the overall amplification process of RNA in thepreparation of RNA vaccine,and verified the anti-tumor activity of the amplified RNA through in vivo and in vitro experiments.The achievements of this study are as follows:1.Confirmed the immunogenicity of purified tumor tissue cell antigen was higher than that of tumor tissue total immunogen.Hepatocellular carcinoma cells were isolated and purified by tissue enzyme digestion and differential centrifugation.Hepatocellular carcinoma cells and hepatocellular carcinoma tissue proteins were obtained by ultrasound.The above two groups of proteins were immunized to Balb/c mice,ELISA was used to determine the immune titer of plasma antitumor antigens in mice,Trizol was used to extract the total RNA of mice spleen,and the integrity and content of RNA were extracted by gel electrophoresis and UV detection.The results showed that:(1)the anti-tumor titer of serum obtained from animals immunized with purified hepatocellular carcinoma protein antigen was significantly better than that obtained from hepatocellular carcinoma tissues;(2)the anti-hepatocellular carcinoma activity of spleen cells immunized with hepatocellular carcinoma protein was higher than that of hepatocellular carcinoma tissues.(3)RNA electrophoresis showed that the extracted RNA was complete.The content of extracted RNA was about 150 ug/spleen and OD260/OD280 was between 1.8 and 2.0.2.Successful establishment of a technique for amplification of immune RNA in vitroThe total RNA of splenocytes obtained from immunized mice was reverse transcribed into cDNA.In the reverse transcription process,(1)Twelve oligonucleotide sequences binding to poly(A)were designed to ensure that all poly(A)mRNA in total RNA were retrieved and a common primer was included for the next step of DNA amplification.(2)Oligonucleotide Cap,including promoter sequence and conversion sequence GGG of T7 RNA polymerase,was designed.For retroviral transcription,all the cDNAs were amplified synchronously by universal primers and long fragment amplification PCR reagents.The amplified DNA was observed by electrophoresis,and the DNA content was detected by ultraviolet detection.Usingamplified cDNA as template,T7 in vitro RNA transcription reaction system was used to transcribe the whole mRNA.The transcribed RNA was detected by ultraviolet detection.GAPDH,Actin,IFG-? were selected,and primers with 179 bp,987 bp,and1424 bp were designed according to the gene sequence.The same volume of anti-tumor iRNA and the whole amplified AT-iRNA were used as templates for real-time reverse transcription quantitative PCR(RT-qPCR)and gel electrophoresis detection.By observing the Ct value and the brightness and location of electrophoretic bands,the integrity of the whole amplified RNA was judged.The effect of RNA amplification was calculated by RNA content.The results showed that(1)4ng reverse transcription DNA was amplified in vitro,and electrophoresis showed that the amplified products of different length fragments were produced.The content of the amplified products was 2.5 ug,and the amplification efficiency was 625 times.(2)The template of transcription in vitro is100 ng of the DNA,and the maximum number of amplifications is 13 ug,more than100 times.(3)The expression of GAPDH and IFN-? was detected by RT-qPCR after reverse transcription using the same volume of anti-tumor iRNA and the whole amplified AT-iRNA as templates.The relative quantitative method was used to calculate the amplification multiples of these genes after overall amplification.The results showed that compared with the original iRNA,GAPDH and IFN-? genes in the amplified AT-iRNA were effectively amplified,with multiples ranging from 200 to1200.This not only demonstrated the fidelity of the in vitro amplification process,but also showed a significant increase in the absolute content of gene mRNA.3.Experiments in vivo and in vitro confirmed that the amplified ribonucleic acid had anti-tumor biological activity.Human peripheral blood mononuclear cells were isolated and obtained in vitro.The amplified ribonucleic acid and non-amplified RNA were respectively applied to peripheral blood mononuclear cells.The stimulated cells were applied to human hepatocellular carcinoma cell lines.The activity of cells was detected by CCK-8.The content of IFN-? in the supernatant of immune cells was detected by ELISA.Animal models of hepatocellular carcinoma cell-bearing mice were prepared.Commercialanti-hepatocellular carcinoma immune RNA and amplified ribonucleic acid(low dose and high dose)were given respectively.The inhibition of tumor growth was judged by observing the weight and volume of tumor cells.The activity of tumor cells was observed by HE staining.The phenotypic changes of lymphocyte were detected by flow cytometry and the apoptotic phase was detected by RT-qPCR.The expression of related genes and the expression of cytokines related to immune cells.The results showed that(1)In vitro experiments showed that lymphocytes stimulated by original iRNA inhibited the growth of tumors by 50.23%,and lymphocytes stimulated by new AT-iRNA inhibited the growth of tumors by 60.08%.There was a significant difference between the new AT-iRNA group and the simple lymphocyte group(p < 0.05),but there was no significant difference between the new AT-iRNA group and the original group.Initial iRNA has the same antitumor effect.Tumor killing experiments in vitro confirmed that AT-iRNA could induce tumor killing of human peripheral blood lymphocyte.(2)IFN-? was effectively secreted in the supernatant of lymphocyte stimulated by iRNA.The level of IFN-? in AT-iRNA stimulation group was significantly higher than that in original iRNA stimulation group.(3)Compared with the tumor-bearing mice and the first-generation nucleic acid group,the tumor volume of the mice treated with 150?g new AT-iRNA decreased significantly in the later period of treatment,and the tumor volume and weight were significantly lower than those of the tumor-bearing mice.(4)HE staining showed significant cell death in the 150?g AT-iRNA group.There was no significant difference in the expression of CD4,CD8.CD56.CD62 L detected by flow cytometry.(5)Real-time quantitative PCR results showed that the relative expression levels of Bcl2,Casp3 in tumor cells of mice treated with 150?g new AT-iRNA were significantly lower than those in the first-generation nucleic acid group.These results suggest that the anti-tumor mechanism of AT-iRNA may be to inhibit the expression level of cancer-related genes,thereby inhibiting the malignant growth of cancer cells.The immune related genes TNF,IFN-?,IL12 A and IL12 B in spleen tissue were significantly down-regulated.These results suggest that the novel AT-iRNA may not kill tumors through immune cells in vivo,and its mechanism needs further study.In conclusion,this research takes the lead in introducing the innovative technology of molecular amplification into the preparation process of anti-tumor iRNA at home and abroad.By combining the innovative and integrated advantages of molecular technology,we take the lead in establishing the preparation technology of anti-tumor immune iRNA based on the innovative and integrated advantages of molecular technology in our country,which ensures the preparation of immunogen,the extraction,amplification and purification of immune nucleic acid,The purity and immunogenicity of tumor cell antigens in immune animals were greatly improved by the specific isolation technology of tumor cells.The geometric series of antitumor active RNA(RNA)was amplified by the whole amplification of anti-tumor RNA in vitro instead of the traditional simple extraction technology.Long-term preservation of patients' individualized anti-tumor immune RNA can obtain highly effective and specific anti-tumor immune iRNA permanent resources.A large number of anti-tumor immune RNA can be obtained from small animals,which greatly reduces the production cost of anti-tumor iRNA.which provides a new method for cancer immunotherapy.