Establishment Of A Mouse Model Of Graves' Ophthalmopathy And Observation Of The Curative Effect Of Pingmu Decoction

Objective:The hTSHR-A subunit recombinant plasmid with electroporation ware used to immunize BALB/c female mice and hTSHR-A subunit activated splenocytes to immunize homologous BALB/c female mice to establish a Graves ophthalmopathy mouse model in each case,and the evaluation was optimized to be more ideal.To establish a mouse model of Graves'ophthalmopathy;and the efficacy of Pingmu Decoction in a mouse model of Graves'ophthalmopathy.Method:Chapter 1:Establishment of a mouse model of Graves'ophthalmopathyThe first part was to establish a Graves ophthalmopathy animal model by immunizing BALB/c female mice with hTSHR-A subunit recombinant plasmid.The second part establishes an animal model of Graves'ophthalmology by using hTSHR-A subunit activated splenocytes to immunize homologous BALB/c female mice.At the end of the experiment,the changes of the eye appearance of the mice wereobserved.The volume of the extraocular muscles in mice was measured by MRI scan.The serum thyroid hormone levels and antibody TRAb levels were detected by ELISA.The histological changes of thyroid and eyelids were observed by pathological staining.The electronic analysis scales weighed the fat tissue and eyeball weight of the mouse eyeball and calculated the percentage.The Graves'ophthalmopathy mouse modelsestablished by the two methods were compared,and the most ideal mouse model of Graves'ophthalmopathy was selected by evaluation.Chapter II:Therapeutic effect of Pingmu Decoction on the mouse model of Graves'ophthalmopathyA preferred mouse model of Graves'ophthalmopathy was constructed in the same way as in Chapter 1.Serum TRAb was detected and positively expressed as a model.They were randomly divided into the following model groups:Pingmu decoction low-dose group(ping low group),Pingmu decoction medium group(pingzhong group),Pingmu decoctionhigh-dose group(pingao group).After 30 days of intragastric administration of Pingmu Decoction in low,medium and high doses,ELISA was used to detect serum thyroid hormone levels and the level of TRAb in mice.The histological changes of thyroid gland and eyelid was observed by HE staining.The weight of the adipose tissue and eyeball after the eyeball of the mouse were weighed by electronic analytical balance and the percentage was calculated.Result:Chapter 1 Establishment of a mouse model of Graves'ophthalmopathyThe first part of hTSHR-A subunit recombinant plasmid immunized BALB/c female mice to establish a Graves ophthalmopathy mouse model.1.At the end of the experiment,in the model group,3/12(25%)of the mice had enlarged eye sockets,chemosis,eyelid edema,conjunctival congestion and exophthalmos.2.MRI scan showed that the extraocular muscle volume of the model group was significantly increased.3.Compared with the blank group(Ctrl)and the empty plasmid group(GV219),the serum FT4 level of the model group was significantly increased(P<0.05),of which 4/12(33.33%)was higher than the upper limit of the normal reference range(The empty plasmid group`x+2S);serum TSH level was significantly decreased(P<0.05),of which5/12(41.67%)was lower than the lower limit of the normal reference range(The empty plasmid group`x-2S);serum TRAb level was significantly increased(P<0.01 or P<0.05),wherein 5/12(41.67%)is above the upper limit of the normal reference range(The empty plasmid group`x+2S).4.Compared with the Ctrl and GV219 groups,the percentage of post-ocular fat and eyeball weight in the model group was significantly higher(P<0.01).5.Model group 7/12(58.33%)mice showed thyroid histological changes,thyroid follicular epithelial cells proliferated,some acinar cavity material absorption,a number of lymphocytes infiltrated,a few follicles destroyed,visible The follicular epithelial cells are papillary and protrude into the cavity.6.Model group showed that 6/12(50%)mice had pathological changes in the eyelid tissue.Behind the eyeball,a large number of fat cells accumulate,the fat cell gap is widened,the muscle fibers are disordered and broken,the collagen fibers are increased between the muscle fibers and between the fat cells,the mucus deposits,the adipose tissue of the muscle space is proliferated and accumulated,and the mast cell infiltration is obvious.The second part of hTSHR-A subunit activated spleen cells immunizes homologous BALB/c female mice to establish an animal model of Graves'ophthalmopathy.1.The eyes of the model group(Implantation of hTSHR-A subunit recombinant plasmid to immunize mice with activated splenocytes 10~6)1/12(8.33%)developed chemosis,eyelid edema.2.MRI scans showed that the extraocular muscle volume of the model group was significantly increased.3.Compared with the control group(Implantation of empty plasmids to immunize mice with activated splenocytes 10~6),the serum FT4 level of the model group was significantly increased(P<0.05),of which 1/12(8.33%)was higher than the upper limit of the normal reference range(control group`x+2S),serum TSH level Significantly decreased(P<0.01),of which 3/12(25%)was lower than the lower limit of the normal reference range(control group`x-2S);serum TRAb levels increased,but the increase was not statistically significant(P>0.05).4.Compared with the control group,the percentage of post-ocular fat and eyeball weight in the mice was significantly increased(P<0.05).5.Model group 8/12(66.67%)mice thyroid epithelial cells became flat due to compression,thyroid follicular epithelial cells had obvious hypertrophy and hyperplasia,accompanied by a number of lymphocytes infiltrating.6.Model group showed that 7/12(58.33%)mice had pathological changes of the eyelids,which were visible after the eyeballs.A large number of fat cells accumulate,the interstitial space of fat cells is widened,the arrangement of muscle fibers is disordered and broken,the collagen fibers of blue staining between muscle fibers and fat cells are increased,the adipose tissue of muscle space is proliferated and accumulated,the infiltration of mast cells is obvious,and there was accumulation of mucus between fat cells.Chapter II Therapeutic effect of Pingmu Decoction on the mouse model of Graves'ophthalmopathyOf the 145 model mice,59 of them had serum TRAb levels above the upper limit of the normal reference range(blank group`x+2S).As model mice,and randomly grouped,medication observation,the results show:Compared with the model group,serum FT4 levels decreased after different doses of Pingmu Decoction,and the Pingzhong group decreased significantly(P<0.05);serum TSH levels increased,and the Pingzhong group was more obvious(P=0.05).);Serum TRAb decreased significantly in each group(P<0.01);the percentage of adipose tissue and eyeball weight in the Pingzhong group and Pinggao group decreased significantly(P<0.05 or P<0.01);the orbital histopathology showed that Pingmutang different dose groups The accumulation of fat cells and the infiltration of lymphocytes in the eyeballs of mice were more than those in the blank group,which was lower than that in the model group.The histopathology of thyroid showed that the substances in the thyroid part of the thyroid gland in different dose groups of Pingmu Decoction were absorbed,and the follicular cavity was uniformly red.The dyed gelatin,interstitial,and a number of lymphocytes infiltrated,the degree of infiltration was more than the blank group,less than the model group.Conclusion:1.hTSHR-A subunit recombinant plasmid with electroporation immunized BALB/c female mice combined established Graves ophthalmopathy mouse model and human Graves ophthalmopathy pathological features and serological expression are closer.2.Pingmu Decoction can effectively reduce the levels of serum FT4 and TRAb in the mouse model of Graves' ophthalmopathy,increase the level of serum TSH to a certain extent,improve thyroid inflammation,and effectively reduce the accumulation of adipose tissue after eyeball.