| Objective: Discuss methods of non-operation modeling of SPID of female rats conforming to clinic manifestation.Dynamically evaluate at multi-time of feasibility,effection and accurately master mechanism of action of non-operation modeling from different angles of morphology,pathology,etiology and immunology;With the aid of syndrome differentiation of Traditional Chinese Medicine,maximum simulated clinic pathogenesis process,classified representation index and determined symptom affiliation of SPID of non-operation rats modeling.Methods: This study contains two parts of experimental research of an animal modeling.Experiment one:The 240 Wistar female rats were divided into 8 groups asrandom,which were No.1 non-operation,No.2 non-operation,non-operation sham,No.1 operation,No.2 operation,an operation sham,an inducement intervened and a natural observation group,with 30 female rats at each group.All employed non-operation methods to modeling in the No.1 non-operation,No.2 non-operation and the non-operation sham groups.Meanwhile,employed operation methods to modeling in the No.1 operation,No.2 operation,and the operation sham groups.The inducement intervened group used only by fatigue and starvation and the natural observation group used without any human intervention in modeling.With accomplishment of modeling,selected three fixed moment and dissected 10 female rats as random at every moment.Observed and recorded variations of the fallopian tubes,ovaries and morphology of(with general morphology and microscopy morphology both)connective tissue,the scores of Pelvic adhesion,the scores of inflammation,the rates of modeling and the positive rates of pathogen by vaginal sampling of dissected female rats of each group.Experiment two:The 240 Wistar female rats were divided into 8 groups as random.The methods of dividing groups,non-operation modeling methods of SPID of female rats and observation moment were all at the same as experiment one.Measured volume of protein secretion of cytokines TNF-?,TGF-? and VEGF of the female rats fallopian tubes,ovaries,connective tissue of pelvic cavity and the serum employed by ELISA.Detected the gene transcription levels of cytokines TNF-?m RNA,TGF-?m RNA and VEGFm RNA of the female rats fallopian tubes,ovaries and connective tissue chose by QPCR.Analyzed the fluctuation of the expressional rate of T cell subset CD3+ and the specific value of CD4+/CD8+ used by flow cytometry assays of peripheral blood in rats.Experiment three: The 120 Wistar female rats were divided into 3 groups as random,which were escherichia coli infected group with 55 rats,mycoplasma infected group with 55 rats and natural observation group with 10 rats.Both of the escherichia coli infected group and the mycoplasma infected group used non-operation modeling.Observed and recorded vital signs,appearance,response of belly pushed,behavior of open field test,secreta of cervix uteri,variation of bite and sup and urine and stool.In virtue of cluster and principal component analysis,made statistic occurrence frequency of the representation symptom of non-operation modeling of the female rats and classification of syndrome.Results: Experiment one: 1.In the aspect of effect of macroscopic modeling the manifestationof rats in general morphology of non-operation group was similar withoperation group at the same moment.At the first moment,there were symptoms appeared as following:avum abdominis congestion,fallopian tubes tumidness and tortuosity,fester attached to fimbriated extremity of fallopian,congestion in several ovaries,tumidness,congestion in the connective tissue of pelvic cavity of the rats.At the second moment,the symptom of tumidness and congestion inthe avum abdominis,the fallopian tubes,ovary and the connective tissue of pelvic cavity of rats had improved with dispersive pus point,a small amount of inflammatory cells infiltrated occasionally were found in the mesenchyme of ovary.And also,the inflammatory cells infiltrated partially were found in the connective tissue of pelvic cavity.At the third moment,there were symptoms appeared as following:adhesion and packaging in the tissue and organism of avum abdominis,fallopian tubes thickening,hydrops,ovarian cyst occasional,hyperplasia the connective tissue of pelvic cavity.Adhesion of avum abdominis only appeared at the third moment in the No.1 non-operation,No.2 non-operation group,No.1 operation and No.2 operation group.The scores of adhesion of avum abdominis were all higher than the natural observation group.There was significant difference(P<0.05).Otherwise,there was no significant difference among groups(P>0.05).2.In the aspect of effect of microcosmic modeling,the manifestation in histopathology of the non-operation group was similar with the operation group at the same moment.At the first moment,the fallopian tubes showed following symptom:the construction of fallopian tubes wall showed vagueness,the mesenchyme was tumidness and congestion,the cell arrangement was disordered and loosen,the part of cilium was short and less and neutrophile granulocyte infiltrated partially.In the meantime,less neutrophile granulocyte was found in mesenchyme of ovary.The connective tissue of pelvic cavity showed hemangiectasis,congestion and neutrophile granulocyte infiltrated partially.At the second moment,the margin of fallopian tubes of rats showed irregularity and the inflammatory cells infiltrated partially were found in each organization of the fallopian tubes.At the third moment,the fallopian tubes of rats showed the following symptom:the area of lumen was enlarged,the pink liquid remained,obstruction,the wall had begun to thin,fibroblast became hyperplasia,the plica mucosa became hyperplasia and fusion,the inflammatory cells infiltrated multilayer,the necrotic epithelial cells and inflammatory cells were found in the gland tubes.In the ovaries of rats,the cystic structure appeared occasionally,with pink liquid filled and no mature follicle in it.For the pelvic cavities of rats,the inflammatory cell of connective tissues infiltrated multilayer and the fibroblast became hyperplasia.In a conclusion,the scores of inflammation of the fallopian tubes and the pelvic cavities connective tissues under the optical microscope of the No.1 non-operation,No.2 non-operation,No.1 and No.2 operation group were no significant difference(P>0.05)at the same moment compared with groups,which were all apparently higher than the natural observed group of the same moment at the first,second and third group,and also,which were apparently higher of the third moment than the first and second moment of the same group(P<0.05).Compared the scores of inflammation under the ovary optical microscope at the three moment above were all approached those of the natural observation group after modeling.There was no significant difference(P>0.05).3.In the aspect of modeling rate,no modeling rats lived at the first and second moment.The rate of modeling for rats was 90%,90%,80% and 80% respectively of the No.1 and No.2 non-operation,No.1 and No.2 operation group at the third moment.There was no significant difference among the groups above(P>0.05).4.In the aspect of pathogens culture,the positive rate of escherichia coli for female rats were 100% and 90% respectively of the No.1 non-operation,No.2 non-operation,and,the positive rate of mycoplasma urealytium for rats was 90% and 90% respectively of the No.1 non-operation and No.2 non-operation group at the first moment,so that there was apparently higher than other groups at the same moment(P<0.05),which tend to decrease thereafter and decreased to the lowest point at the third moment,as a result there was no significant difference compared to the natural observation group at the same moment(P>0.05),and there was apparently lower than the same groups at the first and second moment(P<0.05).Experiment two:1.The volume of protein secretion of cytokines TNF-?,TGF-? and VEGF of femalerats fallopian tubes and connective tissue of pelvic cavity and the serum of the No.1 non-operation,No.2 non-operation group,No.1 operation and No.2 operation group was no significant difference compared among groups at the same moment(P>0.05),which raised during whole process after modeling and all higher than the natural observation group at the same moment,so that there was significant difference(P<0.05),and then it reached to the highest point at the third moment,and there was significant rise compared to the same group at the first and second moment(P<0.05).The volume of protein secretion of cytokines TNF-?,TGF-? and VEGF in the serum of female rats ovaries in 8 groups were all approximately to the natural observation group,therefore,there was no significant difference(P>0.05).2.The expressional rate of T cell subset CD3+ and CD4+/CD8+ of peripheral blood of the No.1 non-operation,No.2 non-operation group,No.1 operation and No.2 operation group was no significant difference compared among groups at the same moment(P>0.05),which decreased during whole process after modeling and all lower than the natural observation group at the same moment,so that there was significant difference(P<0.05),and then it reached to the lowest point at the third moment,and there was significant decreased compared to the same group at the first and second moment(P>0.05).The gene transcription levels of cytokines TNF-?m RNA,TGF-?m RNA and VEGFm RNA of femalerats fallopian tubes,ovaries and connective tissue of the No.1 non-operation,No.2 non-operation group,No.1 operation and the No.2 operation group was no significant difference compared among groups at the same moment(P>0.05),which raised during whole process after modeling and all higher than the natural observation group at the same moment,so that there was significant difference(P<0.05),and then it reached to the highest point at the third moment,and there was significant rise compared to the same group at the first and second moment(P<0.05).The gene transcription levels of cytokines TNF-?m RNA,TGF-?m RNA and VEGFm RNA of the female rats ovaries in the 8 groups were all approximately to the natural observation group,therefore,there was no significant difference(P>0.05).Experiment three: Within the 110 rats of non-operation modeling,there were 93 rats appeared easily irritable,dark red of two ears,dry and lack of saliva of tongue nature and mulberry of tongue color which were concluded as Qi stagnation and blood stasis and about 84.55% of the modeling female rats.The average temperature of the anuses of the female rats raised fastly after modelling,which reached the peak at the 3rd to 7th day,after that decreased gradually,and tended to be stable at the 12 nd to 14 th day.At the 1st to 10 th day,the weights of the female rats slashed,and then those tend towards stable,till the 14 th day,the average loss weights of the female rats were about 15 g compared with the initial average ones after modeling.After modeling,Pushed belly of the female rats in order to irritate them,the amounts of defense acts and the frequency of attack both increased fluctuated.At the 14 th day,the amounts of defense acts raised 1.1 times compared with the 1st day,and also,the frequency of attack raised 1.3 times compared with the 1st day.When the escherichia coli infected group and the mycoplasma infected group went into the open field,the time of lingering at the center position increased apparently,at the same time,the amounts of grids of landscape climbed,the frequency of leaving ground and the frequency of grooming behavior reduced obviously.The differences had statistical significance compared with the natural observation group.Conclusions: According to modeling standard of female rats of SPID(Which should have at least one of clinicopathologic features those included as followed:the fallopian tubes were thickening,obstruction and hydrops,the fallopian tubes and ovaries were adhesion,lump and cyst,the connective tissue of pelvic cavity were hyperplasia and adhesion,so that the method of non-operation modeling of SPID is successful and The rates of modeling can reach up to 80%~90%;Compared the method of non-operation modeling with operation modeling in those aspects of enterocoelia adhesion,T cell expression of peripheral blood(CD3?CD4/CD8),the volume of protein secretion of cytokines TNF-?,TGF-?,VEGF and genetic expression of cytokines m RNA in the serum and tissue homogenate,there were no significant difference.The non-operation modeling of SPID of the female rats had absolute advantage base on the Qi stagnation and blood stasis(84.6%).The non-operation modeling meets the clinic pathogenesis process of the SPID perfectly.This achievement fills the gaps in the field of the study of non-operation animal modeling and the research of TCM syndrome domestic and overseas. |
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