Screening Of Trichinella Spiralis And Mouse Lewis Lung Cancer Cell Related Antigen And The Anti-tumor Effects Of Its Antiserum

Lung cancer is one of the malignant tumors with the fastest increase in morbidity and mortality and the greatest threat to people’s health and life.Tumor biotherapy is considered to be an emerging treatment for cancer,including targeted therapies,immunotherapy,gene therapy,etc.With the development of biotechnology,the studies have showed that some parasitic infections also have anti-tumor effects.In the 1970 s,Trichinella spiralis infection has anti-tumor effects was discovered by researchers.With the continuous development of research,it has been found in recent years that T.spiralis antigens have inhibitory effects on various tumor growth,such as H7402 liver cancer,MCF₇ breast cancer,Sp2₀ myeloma,Hela cervical cancer and A549 lung cancer.The nude mouse tumor model is one of the commonly used animal models in lung cancer research,however,the nude mice are immunodeficient mice,lacking a complete immune system,the occurrence and development of tumors in mice with the normal process of tumors are quite different.The reference value is open to question.The mouse Lewis lung cancer model is another one of the commonly used animal models in lung cancer research.It is established by inoculating LLC cells into SPF grade C57 BL/6 mice,and the tumor-bearing process undergoes a normal immune response in mice.The model is closer to the normal process of tumors and plays an important role in the pathogenesis of lung cancer,drug therapy and biological therapy.At present,the anti-tumor effect of related antigens between T.spiralis and mouse Lewis lung cancer（LLC）cells has not been reported.In this experiment,the cDNA expression library of T.spiralis muscle larvae was screened.The sHSPs gene were obtained and expressed in prokaryotic expression,which associated with T.spiralis and LLC cell.The effect of sHSPs on LLC cells in vitro was detected.The antiserum was prepared with sHSPs to treat tumor-bearing nude mouse model,in order to observe its anti-tumor effect.Through the detection of cytokine levels and immunohistochemistry,the antiserum of Trichinella spiralis sHSPs inhibited the growth mechanism of Lewis lung cancer cells in mice,which provided a reference for further research on the anti-tumor mechanism of Trichinella spiralis and the development of tumor therapeutic drugs.Screening of antigenic genes related to T.spiralis and LLC cells Positive plaques were obtained by screening the cDNA expression library of Trichinella spiralis muscle larvae with positive serum of LLC cell whole protein antiserum,and positive plaques were amplified by PCR.After the PCR amplification products were sequenced and aligned with the gene sequences in Genbank,The antigenic genes related to Trichinella spiralis and LLC cells,Tsp₀9334,Tsp₀7696,Tsp₀9092 and DQ986457.1 were obtained.After their biological analysis,the gene DQ986457.1 was found to encode Trichinella spiralis small heat shock proteins（sHSPs）with multiple hydrophilic regions and predicted antigenic epitopes.Therefore,the gene was selected for the next experiment.The specific primers were designed according to the sequence of T.spiralis sHSPs.The sHSPs gene was amplified by PCR using the cDNA of T.spiralis muscle larvae as a template,and ligated into the expression vector p ET-32a（+）for prokaryotic expression to obtain recombinant sHSPs.The sHSPs were purified by nickel column affinity chromatography,and used to immunize mice to prepare antiserum.The sHSPs were identified by Western blot.The results showed that sHSPs could specifically bind to LLC whole protein antiserum,indicating that sHSPs have good reactogenicity.Indirect immunofluorescence assay was used to identify the sHSPs antiserum.The results showed that sHSPs antiserum and LLC cells could bind.Effects of sHSPs related with T.spiralis and LLC cell on LLC cells in vitro.The release of LDH was analyzed to analyze the effect of sHSPs on the activity of LLC cells.The results showed that the concentration of sHSPs was 50 μg/ml,and the cells were significantly dead when the stimulation time was 24 h.The effect of sHSPs on the proliferation of LLC cells was detected by CCK-8 method.The results showed that the survival rate of LLC cells decreased with the increase of sHSPs concentration.When the concentration of sHSPs was 50μg/ml,the cell survival rate was only 3.3%.This indicates that sHSPs have an inhibitory effect on the proliferation of LLC cells in vitro.The expression of apoptosis-related protein caspase-3 was detected by Western blot.The results showed that sHSPs stimulated LLC cells for 24 hours,and no caspase-3 activation fragment was observed.It is suggested that the inhibitory effect of sHSPs on the proliferation of LLC cells may not be caused by apoptosis of the caspase family.The anti-tumor effects of antiserum prapred with sHSPs related with T.spiralis and LLC cell in vivo.A suspension of LLC cells was subcutaneously inoculated between the forelimbs and the back of C57 BL/6 mice to establish a mouse Lewis lung cancer tumor model.After inoculation of LLC cells for 7 days,the tumor-bearing mice were treated with sHSPs antiserum for 7 days.Then the mouse were sacrificed,the tumors were excised and weighed,the volume of tumors was measured,and the tumor inhibition rate was calculated.The results showed that sHSPs antiserum had the effect of inhibiting tumor growth,and the highest tumor inhibition rate was 69.23%.The expression levels of IL-4,IL-10,IFN-γ and TNF-αafter treatment were detected by ELISA.The results showed that there was no difference between the treatment groups,indicating that the antiserum of Trichinella spiralis sHSPs did not exert anti-tumor by regulating the changes of these cytokines.The expression of VEGF and Bcl-2 in the tumor tissue of each group was detected by immunohistochemical analysis.Compared with the control group,the expression of VEGF gene was not different,and the expression of Bcl-2 gene was significantly decreased.It is indicated that the anti-tumor effect of Trichinella spiralis sHSPs exerts anti-tumor effect by down-regulating Bcl-2 gene.