The Response Of JAK/STAT Pathway On BmNPV Infection And Using Silkworms As Animal Models For Screening Gout Drugs

JAK-STAT pathway plays an important role in many development processes in Drosophila, such as maintaining stem cells status, the development of eyes, the expression of the pair-rule genes of embryo, etc. JAK-STAT pathway could been activated by bacterial and viral infections of insects and involved in cellular immune response through regulating the differentiation and proliferation of blood.As the animal models, silkworm has many advantages in screening gout drugs, such as low-cost, no biological hazards, no conflicts with the ethics, etc. More importantly, the metabolism of uric acid in silkworms was similar with that in human. In order to investigate the response of JAK-STAT pathway of silkworms to virus infection and the feasibility on using silkworms as the animal models for screening gout drugs, we mainly carried out about two aspects of work as follow.1 The response of JAK/STAT pathway on BmNPV infectionJAK-STAT pathway plays a key role in immune response in mammals. JAK-STAT pathway may cause the dysfunction of B-cells and T-cells and serious immunodeficiency when its activity was lost. The JAK-STAT pathway of insects was firstly found in Drosophila. STAT gene exists two spliceosomes both in Drosophila and the Sf9 cell of Spodoptera frugiperda. In the previous study, an ORF of cDNA of BmSTAT whose length is 2202nt has been cloned (named as BmSTAT-S). Based on the E-clone, the other splicing type of BmSTAT has been successfully cloned by RT-PCR (named as BmSTAT- L). Both of these results indicate that BmSTAT gene does have different alternative splicing types. We have known that the length of the ORF of BmSTAT is 2313nt and codes 770aa. The corresponding genomic sequence (28777bp) of the ORF of BmSTAT has been gained by the sequence assembly of the known genome sequence and Gap Fill. The analysis of the introns and exons of BmSTAT indicates that it has 20 exons, BmSTAT-S is spliced by 1-19th exons while BmSTAT-L is spliced by 1-18th and 20th exons. All the borders of introns follow the GT-AG principle except for the 11th intron whose border is GC-AG.In order to investigate the immune response pathway on anti-virus in silkworms, the expression cassette of BmSTAT was introduced into BmN cells by Non-transposable element, and the transgenic BmN cells were acquired by means of Zeocin screening. After being infected by liner Bm-BacPAK6, the results showed that the expression levels of BmSTAT were enhanced by 32.70% and 14.63% in the transgenic BmN cells and the normal BmN cells, respectively, indicating that the JAK-STAT pathway of silkworm responds to the infection of baculovirus. Comparing with the normal BmN cells, the ratio of infected cells by Bm-BacPAK6 was lower in the transgenic BmN cells, and the discrepancy was about 8.28% to 18.02%. Also the expression level ofβ-galactosidase in infected transgenic BmN cells was lower than that in the normal BmN cells, which may be relate with that the level of BmSTAT in the transgenic BmN cells was higher than that in the normal BmN cells. Research consequences lay a good foundation for further addressing the functions of JAK / STAT pathway on the response of baculovirus infection.In addition, we have imported pIZT-STAT into Silkworm eggs by Sperm-mediated method. After incubation, the Silkworm eggs were reared as normal. The silkworms of G0 which can perform green fluorescent have been gained. A specific band of 0.7kb could be amplified by gfp-specific primers in the genome of fluorescent silkworms initially indicating that the fluorescent silkworms we have gained were transgenic silkworms.2 Using silkworms as animal models for screening gout drugsGout which was caused by the accumulation of monosodium urate crystals in the joints is a disease. The high content of uric acid in hemolymph may cause the occurrence of gouty arthritis, the formation of tumor nodules, high uric acid disorders, uric acid kidney stones, kidney disease, etc. In the past few decades, mouse had been used as an mammalian model for hyperuricemia. But, as the animal models for screening gout drugs, mouse has a series problem: High-cost, Biological hazards, Ethical conflicts, etc. What is most important is that the metabolism of uric acid in mouse is very different from that in human. So identification and evaluation of the therapeutic effects of chemicals in an alternative animal model is desirable.In our study, we evaluated the effects of gouty therapeutic drugs on lowering the content of uric acid and inhibiting activity of XOD by using silkworm model. The results showed that the effectiveness of oral administration of various gouty therapeutic drugs to silkworms is consistent with those results for human. The activity of XOD of silkworm treated with allopurinol was obvious lower and declined in a dose-dependent manner than that of control silkworms,while sodium bicarbonate was failed in inhibiting the activity of XOD. The concentration of uric acid in the both hemolymph and fat body were declined by 90% and 95% at six days post-administered with 25 mg/ml of allopurinol, respectively (P<0.01), While the concentration of uric acid in the both hemolymph and fat body were also declined by 81% and 95% at six days post-administered with 25 mg/ml of Sodium Bicarbonate, respectively (P<0.01). Moreover, the epidermis of silkworm administered with allopurinol or Sodium Bicarbonate all became transparence compared with the negative control group. So we suggested that silkworm larva can be used as an animal model for screen and evaluation of gouty therapeutic drugs.