Objective To understand the base of the neuroanatomy underlying the nociceptive syndrome occurring in cervical zygapophyseal joints,in which pain in upper limb was accompanied. Methods The capsules of the C4-T1 zygapophyseal joints of SD rats were immunostained with anti-calcitonin-gene-related peptide(CGRP) antibody,and a modified tricetry labeling was done as following:the fluorescents,fast blue(FB) and nucleus yellow(NY),were injected,respectively,into the right radial nerve and capsules of the zygapophyseal joints of neck,then, the ipsilateral dorsal root ganglia(DRG) were sectioned and the retrogradely labeled sensory neurons in the DRG were observed and photographed,finally,the sections were further immunostained with anti-CGRP antibody,eventually,the neurons in both of the photos labeled by fluorescents and anti-CGRP antibody were comparatively observed and analyzed. Results The nervers fibers containing CGRP distributed throughout the capsules of the zygapophyseal joints from C4-T1 in the rat.Most of these fibers coursed straightly and individually,and some of them anastomosed to each other to form the networks of the nerve fibers.The neurons retrograde labeled by fluorescents were observed in each of the DRG from C5-T1.These neurons shared a labeling populations of FB single(76%),NY single(16%) and FB/YN double labeling(8%) respectively.Moreover, about 40% fluorescents labeled neurons were immunoreacted to CGRP.Conclusion The peripheral processes from a population of sensory neurons that contain CGRP in the cervical DRG simultaneously innervated both the capsules of cervical zygapophyseal joints and the fore limb with the radial nerve.The present results suggest that the nociceptive syndrome of cervical zygapophyseal joints with pain in human upper limb might be the referred pain occurring in the level of the DRG.

OBJECTIVE:To optimize the decoction technology of Zhuanggufang decoction powder. METHODS:Central com-posite design with 2 factors and 5 levels was conducted to design the test,taking water (fold) and decoction time as independent variables,the extraction amount of icariin and extraction yield overall desirability value of as dependent variables,regression equa-tion were fitted;response surface method was used to optimize the decoction technology of Zhuanggufang decoction powder,and was verified. It was compared with the traditional decoction piece effect. RESULTS:The optimized technology was decocted twice with 16-fold water,20 min each time;relative error of predicted and theoretical value by overall desirability value was 2.97%;af-ter decoction powder and piece decocted in the same conditions,the extraction amounts of icariin were 1.2343 μg/g and 1.1324μg/g,extraction yields were 23.73% and 17.84%,respectively. CONCLUSIONS:Central composite design-response surface meth-od optimizing decoction technology is simple and reliable,the optimized decoction technology is stable and feasible,and the decoc-tion capability of Zhuanggufang decoction powder is better than the traditional pieces.

It has been reported that the small type of neurons in the dorsal root ganglion (DRG) play an important role in pain regulation by a presynaptic mechanism via the metabotropic type-B γ-aminobutyric acid receptors ( GABABR ). In order to understand whether the 2populations of the small type of the neurons, peptidergic and nonpeptidergic, in DRG share the same role, immunoflourescent histochemical methods and confocal laser scanning microscope were employed to investigate the expression of the GABABR in the peptidergic and nonpeptidergic small DRG neurons. The results revealed that 92% of the peptidergic and 90% of nonpeptidergic small DRG neurons express GABABR in their perikarya and central processes, which distribute in the various laminae of the spinal dorsal horn. These results suggest both the peptidergic and nonpeptidergic populations of the small neurons in the DRG share similar role in pain modulation via presynaptic mechanisms but in given laminae of the spinal dorsal horn.

Objective To investigate the effect of mice gender on the TSH receptor antibody(TRAb)titers, the levels of TT4,and the degree of thyroid hyperplasia by establishing an animal model of Graves′ disease in male and female BALB/c mice. Methods Male and female BALB/c mice were immunized with recombinant adenovirus expressing TSHRA subunit(Ad-TSHR289)to induce Graves′ disease. Animals were injected 3 times at intervals of 3 weeks. All mice were sacrificed 4 weeks after the last injection to obtain blood for measurement of TSHR antibody titers and TT4evels, and thyroid glands for histological examination. Results TRAb positive rates were 100% both in female or male mice. No significant difference was observed in titers of TRAb between them. The incidence of hyperthyroidism in female mice was higher than that in male mice, being 75.0% and 41.7% respectively. There was statistical difference in levels of TT4between females and males(P＜0.01). Mice with high TT4exihibited marked thyroid hyperplasia. Conclusion Despite TSHR antibodies were similar between female and male mice, the incidence and degree of hyperthyroidism showed sex bias in Graves′s animal model. The results indicated that it was easier to induce model in females than in males by immunizing BALB/c mice with Ad-TSHR289.

Objective To compare the efficacy of Graves disease animal models induced by thyroid stimulating hormone receptor (TSHR) plasmid DNA (pcDNA3.1-TSHR) and by TSHR A subunit recombinant adenovirus(Ad-TSHR289),and to investigate the influence of duration for preparing animal model induced by Ad-TSHR289 on Graves hyperthyroidism and its related indices.Methods The plasmid group and the adenovirus group were set up respectively.The plasmid group:21 female BALB/c mice were randomly divided into model group (n =12) and control group (n =9).The model group were injected intradermally with pcDNA3.1-TSHR 50 μg,once every 3 weeks,totally 3 times.Then 4 weeks after the last immunization,the mice were euthanized to obtain blood for testing TSHR antibody (TRAb),total T4,and thyroid tissue for histological examination.The controls were injected with the same dose of pcDNA3.1 in the same way.The adenovirus group:52 female BALB/c mice were divided into 10-week model group (n =8),14-week model group (n =10) and 18-week model group (n =8),and the respective controls (n =8,n =10,n =8) were set up.All model groups were injected intramuscularly with Ad-TSHR289,three times at three weekly intervals.Then the mice were euthanized at 4,8 and 12 weeks to test TRAb,total T4 level and to observe the change of thyroid histology.The controls were treated with the same dose of Ad-lacz in the same way.Another 8 mice were scheduled to test the dynamic variation of TRAb before and after the 3 times immunization.Results In the plasmid model group,only two of 12 mice developed weak antibody responses against TSHR,and no elevated total T4 level and no hyperplasia changes of thyroid were observed.In the 10-week model group,all mice had high level TRAb [(807.65 ± 136.33)U/L,Six-eighths mice had hyperthyroidism exhibited hyperplasia changes.In the 14-week model group,the TRAb level [(650.12 ± 192.88) U/L]and the incidence of hyperthyroidism (3/10) were lower than those in 10-week group.Histologically,the degree of thyroid hyperplasia lightened to a small extent,but its positive rate did not decline.In the 18-week model group,only 2 of 8 mice displayed slightly elevated TRAb level,and no mice showed increased total T4 level.Additionally,thyroid tissues of 2 mice were mildly abnormal.Compared with the model groups at different time,the change of antibody levels of the mice for TRAb dynamic observation exhibited the similar trend.Conclusions Being good at repeatability and high incidence of hyperthyroidism,the animal model of Graves disease induced by Ad-TSHR289 is still an ideal research tool presently.The duration of model ean be maintained 18 weeks,and 10 weeks is the best period to snstain characteristic of Graves disease.

Objective To investigate the feasibility of inducing neonatal immunotolerance against Graves'disease by gene TSH receptor (TSHR) 289 and its possible mechanism.Methods Neonatal (0-24 h) female BALB/c mice were divided into intraperitoneal injection group,intramuscular injection group,model group,and normal control group.The intraperitoneal group and the intramuscular group were further divided into low-dosage,middle-dosage,high-dosage tolerance groups,and the coresponding control groups.The tolerance groups and the controls were intraperitoneally or intramuscularly pretreated with low-dosage( 1×106 particles),middle-dosage( 1 × 108particles),high-dosage( 1 × 1010 particles)of Ad-TSHR 289 or Ad-lacz respectively.6 to 7 weeks later,the normal control group received intramuscular injection with Ad-lacz; the other groups were immunized with Ad-TSHR289,three times at 3 weeks interval.10 days after the first immunization,serum TRAb was detected.4 weeks after the last immunization,serum TRAb,TT4,splenic CD4 + CD25 + Foxp3/CD4 + were tested,and the thyroid tissues were examinated histologically.Results Ten days after the first immunization,no antibody response against TSHR was detected in the two high-dose tolerance groups,but the TRAb titer in respective controls was significantly higher( P＜0.05 ).4 weeks after the last injection,in high-dose tolerance groups,only 1/10 of mice immunized by intraperitoneal or intramuscular injection elicited anti-TSHR antibody,and no mice immunized intraperitoneally had elevated serum TT4.Two of ten mice challenged intramuscularly showed slightly increased TT4 levels,but the respective controls displayed a strong antibody response( P＜0.01 ) and elevated TT4 level ( P＜0.05 ).The similar percentages of high TT4 and thyroid hyperplasia were found in all groups.Additionally,the frequencies of CD4+CD25 +Foxp3/CD4+in two high-dose tolerance groups were significantly increased as compared to those in controls( P＜0.05 ).The incidence of Graves' disease in the other groups by intraperitoneal or intranuscular injections was not statistically different from those in the corresponding control groups and the model group.Conclusions The immune tolerance against Graves'disease is induced in neonatal mice by either intraperitoneal or intramuscular pathway with specific antigen of TSHR 289,carried by adenovirus vector,and then inhibits Graves' disease in adults. Stimulation with the high-dosage antigen is liable to induce immune unresponsiveness.CD4 + CD25 + Foxp3 +T cells may play an important role in the induction and maintenance of tolerance.

Objective To develop a standard and procedural protocol for the perioperative management of thymectomy for myasthenia gravis(MG) patients and thus to reduce the incidence of MG crisis.Methods From June 1996 to March 2016,466 MG cases received thymectomy we continuously explored key technologies of surgical treatment for MG 466 patients,there were 209 male cases and 259 female cases,with age ranging from 5 to 77 years and chief complaint history ranging from 12 days to 18 years.Symptoms included drooping eyelids,double vision,weakness,shortness of breath,coughing,dysarthria,and difficulties in swallowing and chewing.According to the modified Osserman classification,there were 248 type Ⅰ MG cases,58 type Ⅱa MG cases,66 type Ⅱb MG cases,71 type Ⅲ MG cases,and 23 type Ⅳ MG cases respectively.116 cases received thymecotomy via full sternotomy,204 cases via J type semi-sternotomy,and 146 case via thoracoscopy (including 13 cases via sub-xiphoid approach).Results Perioperatively one case died of sudden death,another patient died of respiratory failure after the second operation for metastatic thymoma,with a mortality rate of 0.42％ (2/466);13 cases had M G crisis (13/466);Six cases underwent tracheotomy (6/466);2 cases had plasmapheresis hypotonic syndrome (accounting for 3.4％ in plasmapheresis cases) and were reoperated to stop bleeding.Postoperatively pathological diagnosis was made,including three thymic atrophy cases,272 thymic hyperplasia cases,178 thymoma cases,and 13 thymic cyst cases.Conclusion A standard and procedural protocol for the perioperative management of thymectomy for MG patients can be developed,which can reduce the morbidity of MG crisis and the incidence of tracheotomy.

Objective To evaluate the treatment efficacy of leukocyte reduction in hyperleukocytic acute myeloid leukemia (HAML) patients with leukostasis grading score (LGS).Methods The data of 54 HAML patients were analyzed retrospectively.The relationship between LGS and leukocyte stasis symptoms or early mortality was observed, and the impact of leukapheresis on LGS was analyzed.Results Among 54 patients with HAML, there were 1 case of M1, 16 cases of M2, 10 cases of M4, 20 cases of M5 and 7 cases of unclassified AML.Based on clinical symptoms and LGS system, 3 cases were LGS 0, 15 cases LGS 1, 17 cases LGS 2, and 19 cases LGS 3.In patients with LGS ≤ 2, the rates of type Ⅰ respiratory failure, central nevers system (CNS) symptoms and early mortality caused by leukostasis were significantly lower than those in patients with LGS 3 (P ＜ 0.05).The LGS of HAML patients was reduced by leukocyte reduction therapy (P ＜ 0.000 1).The LGS of HAML patients treated by leukapheresis and low dose chemotherapy was improved significantly than that of patients treated without leukapheresis (P =0.008).Among 37 cases receiving induction chemotherapy, 20 cases reached complete remission (CR) after the first cycle of induction chemotherapy.CR rate of patients with LGS ≤ 2 was no significantly different compared with that of patients with LGS 3 (P =0.703).Conclusions LGS can be used to evaluate the degree and the improvement status of leukostasis after treatment in HAML patients.The early death often occurres in patients with high LGS.Leukapheresis combined with low-dose chemotherapy can effectively improve the LGS of HAML patients.

Objective To explore the expression and clinical significance of proliferation associated antigen Ki-67 in acute myeloid leukemia (AML). Methods A total of 45 AML patients (including 36 newly diagnosed AML patients and 9 recurrent AML patients) and 20 healthy volunteers (healthy group) were enrolled from October 2012 to January 2016 in Department of Hematology in Fuxing Hospital. The expression of Ki-67 in bone marrow blast cells were detected by flow cytometry (FCM). The relation between Ki-67 level and clinical characteristics, and the prognostic significance of Ki-67 were studied. Results The positive rate of Ki-67 in newly diagnosed AML, recurrent AML patients and healthy controls were (10.38±8.41)%, (20.99± 11.49) % and (40.77±11.97) %, respectively. The positive rate of Ki-67 in newly diagnosed AML patients or recurrent AML patients were significantly lower than that in healthy controls (all P<0.05). The positive rate of Ki-67 in newly diagnosed AML patients was significantly lower than that in recurrent AML patients (P=0.006). The level of Ki-67 in newly diagnosed AML patients did not significantly correlated with age, FAB subtype, white blood cell count, a history of myelodysplastic syndrome (MDS), level of lactate dehydrogenase (LDH), proportion of blats cells, NPM1 gene mutation, FLT3-internal tandem duplication (ITD) gene mutation, chromosome karyotype and response to induction therapy (all P>0.05). There was no significant difference of overall survival between high Ki-67 expression group and low Ki-67 expression group in newly diagnosed AML patients [(780±110) d vs. (788±118) d, P=0.927]. Conclusions The proliferation of blast cells in AML patients is lower than that in healthy controls. Detecting the level of Ki-67 may provide a reference for choosing the cell cycle specific chemotherapy drugs in clinical practice. Monitoring Ki-67 during AML process contributes to monitoring disease progression and predicting recurrence.

Objective To study the expression of domain protein ubiquitin 5(OTUD5)and ring finger protein 186(RNF186)in non-muscle invasive bladder cancer(NMIBC)and their relationship with clinicopathological characteristics and prognostic value.Methods A total of 106 NMIBC patients admitted to Tangshan Traditional Chinese Medicine Hospital from January 2019 to January 2020 were collected.OTUD5 and RNF186 expression in NMIBC cancer tissue and para-cancer tissue were detected by immunochemistry.The differences between OTUD5 and RNF186 expressions in NMIBC cancer tissues with different clinical and pathological characteristics were compared.Kaplan-Meier curve analysis(Log Rank test)was used to analyze the impact of OTUD5 and RNF186 expression on progression free survival of NMIBC.COX regression model analysis was used to analyze factors affecting progression free survival in NMIBC patients.Results The positive rates of OTUD5(62.26％)and RNF186(66.04％)in NMIBC cancer tissue were higher than those in adjacent tissues(11.32％,8.49％),with significant differences(x2=59.146,75.079,all P＜0.05).There was a significant positive correlation between OTUD5 and RNF186 expressions in NMIBC cancer(r=0.659,P＜0.05).The positive rates of OTUD5(72.97％,84.21％)and RNF186(77.03％,86.84％)in T1 stage and high-grade NMIBC cancer tissues were higher than those in Ta/Tis stage(37.50％,50.00％)and low-grade cancer tissues(40.63％,54.41％),and the differences were significant(x2=11.964,13.199;12.143,11.431,all P＜0.05).The 3-year cumulative progression free survival rate of patients in the OTUD5 positive group(40.91％)and RNF186 positive group(45.70％)were lower than that in the OTUD5 negative group(90.00％)and RNF186 negative group(86.11％)(Log Rank test x2=24.710,14.586,all P＜0.05).OTUD5 positive(OR=1.446,95％CI:1.086～1.925),RNF186 positive(OR=1.579,95％CI:1.132～2.024),Tumor TNM stage T1(OR=1.582,95％CI:1.219～2.054)and high pathological grade(OR=1.570,95％CI:1.188～2.074)were independent risk factors affecting progression free survival in NMIBC patients(all P＜0.001).Conclusion The increased expression of OTUD5 and RNF186 in cancer tissue of NMIBC patients are related to tumor TNM staging and pathological grading and are independent risk factors affecting the prognosis of NMIBC patients.