Development Of Melittin-based Gene Therapy Method To Treat Ra

Background:Rheumatoid arthritis (RA), known as an autoimmune, is a serious of symptoms including chronic, symmetric, multi-articular synovial lesions of arthritis. Bee venom used for treatment of RA has been recorded for more than one millennium. "Huangdi Neijing" recorded that bee venom was therapeutic but its toxic to human being. Bee Venom taste spicy, bitter, and have a function of enhancing blood circulation, reducing swelling and pain, relieving rheumatism. Clinical and laboratory data confirmed that bee venom were not only for the treatment of human RA, but also has a good effect on Freund's adjuvant, II collagen and other animal models of RA. Bee venom has very complex compositions, containing a variety of activating peptides. 50% of dry weight of bee venom is melittin, indicating that melittin may be the main active ingredients of bee venom. First, this study was to confirm the efficacy of melittin treatment of RA, and then melittin coding gene was recombined into AAV for melittin based gene therapy for RA. All these works help to develop a new method to treat RA. Part I: Exploration joint protective effect on of melittin against RA animal modelsObjective: to explore the protective effect of melittin on animal models of RA.Methods: 60 Male SD rats were randomly divided into venom group (Bv), melittin group (Me), saline control group (Mn) and the sham group (Nn). The first three groups was injected with complete Freund's adjuvant in the right hind foot palm to induce RA model. Bee venom (1mg/kg), melittin (0.5mg/kg), saline (10ul ) and saline (10ul) were administered in the affected side of Zusanli to treat RA. The experiment was concluded three weeks. body weight, thickness of joint, withdrawal latency of the hind paw were regularly detected after modeling Fos was detected with immunohistochemistry; X-ray was used to find the change of bone and joint. Content of IL-10, TNF-αin joint homogenate were determinate with ELISA.Results: The weight of all rats increased with time, differences are not significant at each time point. Joints were seen swollen at 4d after injection of complete Freund's adjuvant, however no significant change was observed untill 12 days. Me group (0.73±0.09cm), Bv group (0.75±0.07cm) was significantly lower than that of Mn group (0.99±0.06cm) in the thickness of articular by analysis of variance (P <0.05). This trend continued to the end of the experiment. Withdrawal latency of the hind paw in each group were basically the same at the beginning of the experiment, but significance after modeling was observed (day 6) with Me (9.5±0.5S), Bv (10.6±0.6S), Nn (9.8±0.8S) that were longer than in Mn group (6.3±0.6S), analysis of variance showed significant differences (P <0.05). This trend continued until the end of experiment. Fos-positive neurons in Bv, Me, Mn, Nn group of the superficial numbers were 37±6, 34±5, 56±8, 35±4 respectively. Analysis of variance showed that numbers of Fos-positive neurons in Bv, Me, Nn Group were lower than that in Mn group. Melittin and bee venom therapy can improve the performance of paws joint X-ray and inhibit ipsilateral articular secretion of TNF-α, increase the content IL-10. Bee venom and melittin treatment had no effect on the contralateral paws in joint thickness, withdrawal latency and joint cytokine level.Conclusions:1. Melittin is the active ingredient in bee venom treatment of RA;2. Melittin could inhibit the RA joint expression of Th1 type cytokines, and could increase expression of Th2 type cytokines.Part II: Recombination of melittin into adeno-associated virus and its identification in vitroObjective: Recombination of Melittin into adeno-associated virus and its identification in vitroMethod: PCR was employed to amplify of complementary primer containing PmacI point in its 5'end, Mel-cDNA sequence, and BamHI in its 3 'terminator. Mel-cDNA gene sequence synthesized and complemented with Klenow fragment was connected into the T vector. Restriction enzyme digestion and sequencing were used to identificatie its rightness. T vector pGEM-T Easy/NT4 was double digested with PmacI and BamHI, and large fragments was gathered and connected with Mel-cDNA sequences. The new vector was double digested with EcoRI and BamHI, small fragments was collected and inserted into rAAV vector pSSCMV. Combined with pAAV-Ad and pFG140, the three plasmids were transfected into 293 cells, titer was identified with hybridization method. HELA cells were transfected in vitro and melittin in vitro expression was determinatied with immunohistochemical methods.Results: Outcomes of electrophoresis, restriction enzyme digestion and sequencing confirmed that Mel-cDNA sequences was inserted into T vector; pGEM-T Easy/NT4 and Mel-cDNA sequences and restriction site are properly connected. Expression cassette was properly inserted into the rAAV vector pSSCMV; titer of rAAV/Mel was about 1012/ml. In vitro transfection experiment confirmed that melittin can be secreted after transfected with rAAV/Mel.Conclusions:1. EcoRI-NT4-NaeI-TAT-6His-PmaCI-Mel-BamHI expression cassette was constructed;2. Construction of the rAAV / Mel, and melittin can be detected in vitro after infected into HELA cells.Part III: Exploration of anti-rheumatic effect of recombinant adeno-associated virus/melittinObjective: To explore the anti-rheumatic effect of melittin recombined adeno-associated virus.Method: complete Freund's adjuvant induced RA models were used as mentioned in the first part, melittin, rAAV/Mel, rAAV/GFP were given in the Zusanli of affected side to confirm their therapeutic effect. While rAAV/Mel, were injected to different site to find out that whether the therapeutic effect depending on the injection site. Rats were regularly weighed, and thickness of paws joint, withdrawal latency of the hind paw were regularly detected, X-rays was used to find the difference before or after treatment. TNF-α, IL-10 levels in joint homogenate were measured.Results: Joints were seen swollen at 4d after injection of complete Freund's adjuvant, however no significant change was observed untill 12 days. Me group (0.75±0.07cm), RMZ group (0.72±0.09cm), RMZ (0.72±0.04cm), RMT (0.71±0.04cm), RMP (0.65±0.06cm) were significantly lower than RMD group (0.97±0.05cm) and the RC group (0.94±0.04cm) in thickness of paws joint by analysis of variance (P <0.05). This trend has been continued until the end of experiment. Withdrawal latency of the hind paw of each group were basically the same before modeling, it was changed at the first observation (day 6). Me group (10.4±1.1S), RMZ group (9.7±1.5S), RMP group (9.8±1.3S), RMT group (9.6±0.8S) were longer than RMD group (6.2±1.2S) by analysis of variance (P <0.05). This trend has continued to end of the experiment. Recombined AAV with melittin can inhibit the secretion of TNF-αin Me group (78.7±39.2pg/ml), RMZ group (78.3±29.7pg/ml), RMT group (62.3±34.5pg/ml) and the RMP group (68.1±32.5pg/ml), which were higher than that of RC (270.5±23.1pg/ml), RMD Group (328.2±67.3pg/ml). Analysis of variance showed significant differences (P<0.05). While TNF-αin the contralateral joint showed no significant differences. The content of IL-10 in RC, RMD ipsilateral joint homogenates were 21±8.1pg/ml, 24±6.3pg/ml, and Me, RMZ, RMT, RMP Group were 51.2±7.1pg/ml, 53.4±8.0pg/ml, 49.8±7.1pg/ml, 48.2±7.9pg/ml, variance analysis showed that the IL-10 content in RC, RMD ipsilateral joint homogenate were significantly lower than that in the RMZ, RMT, RMP group. Changes of X-ray in Me, RMZ, RMT, RMP group paws joint is better than RC and RMD group. Weight of each group at each time point was not found statistically different.Conclusions:1. Identification of the efficacy of gene therapy with melittin;2. Efficacy of treatment of rAAV/Mel was confirmed with secretion TNF, IL-10.3. By regulary weighing body weight, rAAV/Mel has no side-effect on animals, suggesting that the security of rAAV/Mel treatment to RA.