Experimental Research Of The Treatment Of Beta-Endorphin On CCI Rats Expressed By Recombinant Low-toxic Adenovirus

【BACKGROUND&OBJECTIVES】The application of gene therapy is getting advanced in medical field over the development of application technology.Gene delivery vector is a critical factor in gene therapy system.Although adenovirus infects human,the related pathology is little.Its features showing in gene therapy make it a promising vector.Till now,adenoviral vector gained a great proportion in the basic research and clinical research of gene therapy.The third generation adenoviral vector has been developed.The shortcoming of adenoviral vector showed in gene therapy made the vector not suitable for some specific diseases, such as genetic disorders and chronic pain.Compared to the first generation adenoviral vector,the DNA capacity and expression duration and the lower immune response are the main advantage of the third generation(HD-Ad),which made the relevant research hot in the field of gene therapy.Transgens carried by adenoviral vector could be expressed at a higher level,but the gene delivery vector is not the unique determiner of the expression of interest genes,whose expression can also be affected by the promoter of genes.The therapeutic protein in this project needs to be excreted outside of cells to be transported to the target site and shows their active function.The polypeptide chain translated from the fusion gene should be guided by the signal peptide.So,the adopted sequence of signal peptide in the fusion gene is another important factor affecting the expression level.Chronic pain has been being a big problem to doctors and patients.It is an important topic for research workers to efficiently to relief the pain.A lot of findings proved that the method of gene therapy can be applied in the treatment of chronic pain.When the traditional clinical treatment attenuates the pain,its side effects become the extra suffering, which the current hot gene therapy makes the past.Beta-endorphin is an important component of endogenous analgesic system,which is critical in the analgesic mechanisms. Endorphin can affect the signal transduction pathway which is in charge of passing the nociceptive signals to superspinal center to form the sense of pain.Some research works showed that beta-endorphin can relief chronic pain,but those studies are not sufficient.On the base of previous study,the research about the low-toxic recombinant adenovirus and beta-endorphin is continued.The therapeutic effect of transgenes on the model of chronic pain was discussed,which will promote the application of gene therapy for chronic pain.【METHODS】1.After gaining the human genome,two DNA fragments,the signal peptide of human nerve growth factor and human beta-endorphin,was fused by SOE-PCR method,and the sequence,which can be recognized by FURIN hydrolysis enzyme,was inserted between them to make the secretory expression of fused protein possible.After the DNA sequence was proved right by DNA sequencing method,the expressing fragment was cloned into pCDNA3.1(+) to make pCDNA3.1(+)-HNE expressing human beta-endorphin.At the same time,pCDNA3.1(+)-NEP containing the signal peptide from mouse was made to express human beta-endorphin.Two expressing vectors were transfected into cultured NIH3T3 cells by liposome transfection method respectively.Seventy two hours after transfection,the culture media containing the expressed protein was collected to determine the concentration of beta-endorphin by RIA.The efficiency of two kinds of signal peptides was compared to introduce the secretory expression of protein.2.Two FRT sites was inserted into the adenoviral shuttle vector before and after the packaging signal sequence with the same direction.The shuttle vector pSH-2FRT was linearized with PmeI.And then,the linearized pSH-2FRT transformed the competent cells of BJ5183-AdEasy-1.The plasmid vector for the first generation adenovirus,pAd-HELPer, was generated by the recombination of pSH-2FRT and pAdEasy-1 in BJ5183-AdEasy-1 cells.The genome of helper adcnovirus was released by PacI and transfectcd HEK293 cultured in 6-well plate to package adcnovirus.Ten days later,the helper virus was cropped. It was identified by PCR that there is no RCA adenovirus in the viral stock.The helper adenovirus was propagated for large scale and was tited.3.The DNA fragment of fusion gene was cloned into the main frame vector to generate adcnoviral plasmid vector pST-HNE.The genome of recombinant adcnovirus was released by PmeI and was purified.After the HEK293F cells was transfected by the viral genome,the produced helper virus Ad-HELPer infected the transfected HEK293F cells. Two or three days later,low-toxic adenovirus was packaged and was used to make a large scale propagation with Ad-HELPer.The cells containing propagated virus were cropped and was coped with by melt-thaw method for three cycles to free the virus.The raw virus stock was made after centrifuging and was purified by cesium chloride gradient centrifugation.The virus liquid was desalted with PD-10.Those indices,the titer of low-toxic adenovirus and helper adenovirus,was determined by real-time quantitative PCR. RCA adenovirus was checked at the same time.The proportion of helper adenovirus was calculated.4.The cultured NIH3T3 and HEK293 cells was transfected with generated recombinant virus according to MOI equal to five.Forty eight hours later,the total RNA of cells was extracted and then was transcribed to cDNA.The mRNA from fusion genes was checked by common PCR.NIH3T3 cells were cultured in 6-well plate with sterilized cover glass in each well.The cover glasses with cells were collected after the cells were infected by virus for 48h.After the cells was rinsed and fixed with 4%paraformaldehyde,the cellular immune fluorescent staining was made to check the expression and distribution of beta-endorphin.The culture media was collected at each time point to measure the concentration of beta-endorphin by RIA method to deduce the trend of expression,after the cultured NIH3T3 cells were infected by two kinds of virus.5.The CCI rats models were build up,after which,two different recombinant adenovirus was injected into subarachnoid space at the lumbar fragment.The radiation thermal PWL of experimented animals at right rear feet was determined at several settled time points to analyze the trend of PWL.The cerebrospinal fluid of animals was got to determine the concentration of beta-endorphin by RIA to analyze the trend of target protein. The anesthetized animals was perfused with 4%paraformaldehyde liquid to get the spinal cord tissue after those animals was injected with recombinant virus.The tissue immune fluorescent staining was done to observe the situation of infection by virus and the target protein expression in spinal cord.The dependability between the threshold of PWL and the concentration of beta-endorphin in cerebrospinal fluid was analyzed by linear regression analysis.【RESULTS】1.Two eukaryotic expression vectors,which contain the signal peptides from human or mouse nerve growth factor respectively to introduce the secretory expression of beta-endorphin,were successfully constructed.There was beta-endorphin detected in the culture media of NIH3T3 cells transfected by those two expression vectors,which suggested that these two signal peptides could implement the secretory expression of fusion genes.The results showed there was significant difference between the level of expression product of two vectors,pCDNA3.1-NEP and pCDNA3.1-HNE (p＜0.05) and between them and the blank control pCDNA3.1(+)(p＜0.01),which suggested that the efficiency of secretory expression of two signal peptides was statistically different and the efficiency of signal peptide from human is about 50%higher than that of from mouse.2.The helper virus Ad-HELPer was successfully generated and no evidence of containing RCA adenovirus was obtained by PCR to virus stock.The propagated helper virus was purified and was tited to be 3.67E+9 PFU/ml.3.Low-toxic adenoviral plasmid vector pST-HNE was generated exactly.Low-toxic adenovirus HDAd-HNE was packaged after HEK293F cell were transfected by pST-HNE with the infection of helper virus Ad-HELPer.With co-infection of the helper virus, HDAd-HNE was propagated in large scale.The propagated virus was purified by cesium chloride gradient centrifugation method,and then was desalted.The titer of final virus stock was determined to be 2.59E+12 genomes/ml by real-time quantitative PCR and the proportion of helper virus was 0.16%.There was no RCA adenovirus detected.4.The relevant mRNAs were indirectly detected in those virus-infected NIH3T3 and HEK293 cells.Forty eight hours after NIH3T3 was infected by recombinant virus,cellular immune fluorescent staining of those cells showed that,the fusion genes in HDAd-NHE and Ad-NEP could be expressed and the expression dispersed in the cytoplasm,not cell nucleus.Beta-endorphin could be detected in the culture media from different time points. The concentration of beta-endorphin was relatively good at the first day after infection;the concentration at the third day was higher than that at the first day and the concentration at the seventh day was the highest among three time points.There were significant difference between them and the blank control(p＜0.01).The concentration from Ad-NEP-infected cells was significantly higher than that from HDAd-HNE-infected cells(p＜0.05).5.After the CCI rats models was successfully set up,two kinds of recombinant adenovirus was injected into the lumbar subarachnoid space of CCI rats.The tissue immune fluorescent staining of spinal cord three days after virus injection suggested that those fusion genes in recombinant viruses did express in the cells of spinal pia mater.There was no effect on the basic pain threshold of transgene-expressed beta-endorphin.Ad-NEP-/HDAd -HNE-expressed beta-endorphin in the cells of spinal pia mater was transported into cerebrospinal fluid and the concentration of beta-endorphin reached the peak at the third day,then descending during the following stage or maintaining till the end of observation.A significant difference existed between them and the blank control(p＜0.05).The PWL of rear paw in virus-injected rats was changed significantly(p＜0.05),but because of the descending of beta-endorphin from Ad-NEP,the PWL of that group is close to the pre-therapeutic level.The linear correlation showed that there is dependability between the change of pain threshold and the concentration of beta-endorphin,which suggested that the changing PWL of CCI rats was caused by the recombinant-expressed beta-endorphin.【CONCLUSION】 The efficiency of those signal peptides from human nerve growth factor and mouse to introducing the secretory expression of protein is significantly different,and the efficiency of human signal peptide is better than that of mouse signal peptide.The helper adenovirus played an assisting role in production of low-toxic recombinant adenovirus,which was packaged.The in vitro and in vivo research showed that low-toxic recombinant adenovirus and first-generation adenovirus could express beta-endorphin,which was transported outside of infected cells.But the expression level of low-toxic adenovirus is lower than that of the first generation adenovirus.The expressed beta-endorphin was biologically active and had a therapeutic function,changing the pain threshold of CCI rats.