Cholangiocarcinoma is a malignant tumor with biliary epithelial features.The early diagnosis of cholangiocarcinoma is currently difficult and the treatment outcomes are poor.Its microenvironment includes abundant fibrotic mesenchyme and a variety of cell types,which promote the development and metastasis of cholangiocarcinoma by interacting with tumor cells through mechanisms such as facilitating migration,suppressing the immune response,and inducing angiogenesis and lymphangiogenesis.Immunotherapy is an important tumor treatment approach,and immunotherapy for cholangiocarcinoma has made some progress.This article reviews the characteristics of the immune microenvironment of cholangiocarcinoma,its relationship with immunotherapy,and cutting-edge therapeutic strategies.

ObjectiveThis study aimed to evaluate the clinical efficacy of Ruyi Zhenbaowan（RYZBW）in the treatment of initial and early knee osteoarthritis （KOA） through a prospective multicenter，randomized，double-blind，and placebo-parallel controlled trial. MethodFrom October 13^th， 2021 to December 25^th， 2021， 240 KOA subjects meeting the acceptance criteria were enrolled in 15 sub-centers including Wangjing Hospital， Chinese Academy of Chinese Medical Sciences， and they were randomly divided into observation group and control group， with 120 cases in each group. The intervention measures for the observation group were RYZBW + health education， and the intervention measures for the control group were RYZBW placebo + health education. The intervention period in both groups was four weeks， and they were followed up for four weeks after the intervention. The primary outcome measure was the total score of Western Ontario and McMaster University Osteoarthritis Index score （WOMAC score）， and the secondary outcome measures were the response rate of visual scale （VAS） pain score， WOMAC sub item scores （joint pain， joint stiffness， and joint function）， quality of life （SF-12） score， and traditional Chinese medicine （TCM） syndrome score. Result（1） Efficacy evaluation. The marginal model results showed that the observation group was better than the control group in improving the WOMAC total score and WOMAC pain score in the treatment of KOA with RYZBW， and the difference was statistically significant （P<0.05）. There was no significant difference between the two groups in improving VAS score response rate， WOMAC function score， WOMAC stiffness score， SF12-PCS （quality of life-physical health） score， SF12-MCS （quality of life-mental health） score， and TCM syndrome score. （2） Subgroup analysis. ① In terms of VAS score response rate， the response rate of the observation group was higher than that of the control group for subjects with baseline VAS score of （4， 5］， and the difference was statistically significant （P<0.05）. ② In terms of TCM syndrome score， for subjects aged ［56， 60］ and ［61， 65］， the decrease in total TCM syndrome score in the observation group was better than that in the control group， and the difference was statistically significant （P<0.05）. ConclusionTibetan medicine RYZBW has good clinical efficacy in improving WOMAC total score， VAS score response rate， WOMAC pain score， WOMAC function score， and TCM syndrome score for patients with initial and early KOA， which can fill the lack of Tibetan medicine RYZBW in the treatment of KOA and make a demonstration study for the inheritance and development of ethnic medicine.

Objective To study the effects of Guizhi Shaoyao Zhimu Decoction on factors related to bone destruction and bone protection in rats with collagen-induced arthritis(CIA)based on osteopro-tegerin(OPG)/receptor activator of NF-κB ligand(RANKL)/receptor activator of NF-κB(RANK)signaling pathway.Methods According to the body weight,60 female Wistar rats were randomly di-vided into the following six groups:the normal group,the model group,the Triperygium wilfordii mul-tiglucoside group(0.01 g/kg),the Guizhi Shaoyao Zhimu Decoction low-dose group(8.6 g/kg),the Guizhi Shaoyao Zhimu Decoction medium-dose group(17.2 g/kg),and the Guizhi Shaoyao Zhimu Decoction high-dose group(34.4 g/kg)(n=10 rats per group).The rats in all groups except for the normal group were given 100 μg bovine type Ⅱ collagen on the 1st and 8th days to establish the CIA model,and was injected into the left foot sole and tail root of the rats.After the successful modeling,the rats were treated by gavage for 4 weeks.The general state,body weight,and arthritis index(AI)score of rats were recorded,and the contents of RANKL and OPG in rat serum were determined by enzyme-linked immunosorbent assay.The mRNA and protein expressions of RANKL,RANK,and OPG in the ankle joint were determined through real-time PCR and Western blotting,respectively.Results Com-pared with the normal group,the general state of the model group was poor,the toe swelling was obvious,the AI score was increased,the serum RANKL content was increased,the serum OPG content was de-creased,and the mRNA and protein expressions of RANKL and RANK in the ankle joint were increased(P＜0.05).Compared with the model group,the degree of toe swelling and the AI score of rats in the Guizhi Shaoyao Zhimu Decoction low-,medium-,and high-dose groups were decreased,the serum RANKL content was decreased,the serum OPG content was increased,the mRNA and protein expres-sions of RANKL and RANK in the ankle joint were decreased,the mRNA and protein expressions of OPG were increased,and the RANKL/OPG ratio of the ankle joint was decreased(P＜0.05).Conclusion Guizhi Shaoyao Zhimu Decoction can improve the destruction of joint bone in CIA rats,and its mecha-nism of action may be related to reducing RANKL level,reducing RANKL/OPG ratio,and regulating bone balance.

Objective: To explore the effectiveness of spinal guidance exercise on adolescents of mild idiopathic scoliosis, so as to provide an experimental reference and method for promoting adolescents spinal health. Methods: From the scoliosis screening, 96 children who met the diagnostic criteria were selected in June 2021, and were divided into the experimental group and the control group, with 48 cases in each group. The experimental group received a 12 week Longji Daoyin exercise training, and the control group received health education, observation and monitoring. Before and after the experiment, the axial trunk rotation (ATR) and scoliosis Cobb angle of the two groups were tested respectively, and the subjective sensation of the patients was measured by Scoliosis Research Society-22(SRS-22) questionnaire. Results: After 12 weeks, the ATR and Cobb angles of the experimental group decreased [( 5.02± 2.85，7.59±3.94)°]，compared to (7.08±2.84)° and (15.84±2.00)° before intervention( t =14.02, 17.09, P < 0.01 ). Scores of functional activity, self image and mental health in the SRS-22 score increased from (4.65±0.14)(3.49±0.21) and (3.99±0.17) to(4.77±0.14)(4.62±0.13)(4.42±0.13)( t =-4.86,-39.12,-13.92, P <0.01 ). The ATR and Cobb angles of the control group increased from (6.94±2.15)° and (15.82±1.76)° to (8.25±2.49)° and (18.63±3.12)°( t =7.44,8.99, P <0.01), and the scores of self image and mental health in the SRS-22 score decreased from (3.49±0.19) and (4.00±0.15) to (3.44±0.18) and (3.94±0.14)( t =-3.74,-4.39, P <0.05). Conclusion Early adolescent idiopathic scoliosis is easy to aggravate during the observation period. Longji Daoyin exercise is of preventive and therapeutic significance.

Objective To investigate the adhesion levels in uropathogenic Escherichia coli with various degree of drug resistance.Methods One hundred strains of Escherichia coli isolated from urine specimen were collected from patients admitted to 4 Grade A tertiary hospitals in Tianjin during March 2012 to October 2015.Escherichia coli were divided into drug sensitive group and drug resistant group by drug sensitivity tests with 50 strains in each group.The expressions of fimH,fimA,fimB genes of type I fimbriae and papA,papB,papC,papGII genes of P fimbriae were detected by polymerase chain reaction(PCR)and real-time fluorescence quantitative RCR (RT-PCR),respectively.Adhesion ability of type I fimbriae and P fimbriae were tested by yeast cell adhesion test and erythrocyte agglutination test.Chi square test and t(Z) test were used to analyze the data.Results The positive rate of papGII in drug resistant group (42.0%) was significantly higher than that in the drug sensitive group (16.0%)(χ2 =8.208,P <0.05),while there were no significant differences in the positive rates of fimH,fimA,fimB,papA,papB and papC genes between two groups(all P >0.05).The expression levels of fimH,fimB and papC genes in the sensitive group were higher than those in the resistant group(Z =3.427,t =5.182 and 8.120,all P <0.05).The adhesion ability of strains carrying type I fimbriae in sensitive group was stronger than that of resistant group (χ2 =5.769,P <0.05).Conclusions The decline in adhesion ability of type I fimbriae in drug resistant E.coli strains is possibly associated with the adaptive cost of bacteria,the transcription and deficiency of other genes encoded by fim and pap gene cluster will also affect the adhesion function of type I pili and type P pili.

BACKGROUND:Currently, vertebral compression fractures are the most common osteoporotic fracture in postmenopausal women;however, incidence of refracture has aroused increasing attention due to a lack of standard treatment. OBJECTIVE:To evaluate whether vertebroplasty combined with anti-osteoporosis treatment can reduce refracture rate fol owing osteoporotic vertebral compressive fractures. METHODS:Eighty-nine patients with osteoporotic vertebral compressive fractures undergoing vertebroplasty were divided into control group (n=38) and treatment group (n=51) after making an informed choice about treatment. Chest/lumbar X-ray and bone mineral density determinations were performed through outpatient or inpatient fol ow-up. The spinal stability, bone mineral density and refracture rate of patients in both groups were fol owed up. RESULTS AND CONCLUSION:Seventy-eight patients achieved complete fol ow-up (ranged from 6-39 months, average 26.73 months). There was no significant difference in the spinal stability between both groups (P>0.05), while rare bone trabecula was found in the control group. There was a significant difference in bone mineral density between both groups at postoperative 12, 24, and 36 months (P<0.05). The refracture rate in the treatment group was significantly lower than that in the control group (P<0.05). Our results indicate that anti-osteoporosis treatment can effectively reduce the incidence of refracture after vertebroplasty in patients with osteoporotic vertebral compressive fractures, and this study found satisfactory short-and medium-term clinical outcomes.

OBJECTIVE To investigate the role of gonadotropin-releasing hormone (GnRH) in es-tradiol(E2 ) induced advance of puberty onset in rats. METHODS Postnatal day 18 SD rats were given a daily intragastric administration of corn oil or E2(50 μg.kg-1 ) for consecutive 5 d. The day of vaginal opening (VO), pathological changes in ovary and protein expression levels of GnRH, G protein-coupled receptor 54 ( GPR54) and phospholipase C ( PLC) in hypothalamus were observed. RESULTS As compared to corn oil controll group, VO was advanced by about 12.2 d, corpus luteum was observed in the ovary section, and the protein expression levels of GnRH,GPR54 and PLC in hypothalamus were significantly increased by 47%, 55% and 56% in E2 group, respectively. CONCLUSION E2 induced onset of puberty advance may be closely related to regulation of the expression of GnRH, GPR54 and PLC in hypothalamus.

BACKGROUND:To grasp the optimal multiplicity of infection (MOI) and the time when stronger fluorescence intensities produce can lay the foundation for tracing observation of human bone marrow mesenchymal stem celsin vivo in animal models. OBJECTIVE:To investigate the feasibility of HIV-1 lentivirus carrying enhanced red fluorescent protein to transfect human bone marrow mesenchymal stem cels. METHODS:Human bone marrow mesenchymal stem cels at passage 4 were divided into blank group and MOI 2, 3, 4 groups. After that, the cels were seeded into 12-wel plates at a density of 5.0×105 , and cultured in 1 mL complete medium for adult bone marrow mesenchymal stem cels containing 1% fetal bovine serum. The infectious titer of lentivirus-carried enhanced red fluorescent protein was adjusted to 1.0×10 11 TU/L. Lentivirus solution 10, 15, 20 μL at MOI=2, 3, 4 were respectively added into the MOI 2, 3, 4 groups, and 10μL PBS was added into the blank group. At 24 and 72 hours after transfection, the expression of red fluorescence was observed under an inverted fluorescence microscope and the transfection efficiency was calculated. RESULTS AND CONCLUSION:Enhanced red fluorescent protein expressed stably in bone marrow mesenchymal stem cels. At 24 hours after transfection, red fluorescence could be seen under the inverted fluorescence microscope and achieved the peak at 72 hours after transfection. Within 21 days after transfection, there were no differences in the number of human bone marrow mesenchymal stem cels between the MOI 2, 3, 4 groups and blank group (P > 0.05). These results show that the HIV-1 lentivirus carrying enhanced red fluorescent protein is feasible to transfect human bone marrow mesenchymal stem cels, with the highest transfection efficiency when the MOI=4, which can express at least for 21 days and have no effects on the proliferative activity of labeled cels.

Objective To examine the detection rate of 30 known virulence factors (VFs) of extraintestinal pathogenic Escherichia coli(ExPEC), and to investigates the epidemiology of ExPEC in elderly nosocomial infection. Methods A to?tal of 140 ExPEC clinical isolates from elderly nosocomial patients in hospitals in Tianjin were investigated. Multiplex PCR was performed to detect the 30 virulence factors among the E.coli strains and the detection rate of virulence factors for Ex?PEC were compared between isolates from different sites of infection.Fifty E. coli strains were shown to carry fimH gene that was amplified and sequenced. These sequences were used besides3 references strains (CFT037、UTI89 and K-12 ) to detect SNPs of fimH gene using DNAMAN Version 6.0.3.93 these 53 fimH sequences were used for genotyping and building dendrogram by MEGA4 software. Results In ExPEC, the following virulence factor genes, fimH, traT, fyuA, iutA and kpsMT II, had a higher detection rate than those of the rest . The following virulence factor genes, kpsMT II, K5, papC, pa?pEF ,papG allele II (Internal), papA, cnf1 (CNF), sfa/focDE and rfc had a a higher detectionrate from non-urine origin sam?ples than those from urine origin samples. fimH SNPs analysis of the 50 clinical isolated samples and 3 references samples showed 60 SNPs at 57 polymorphic sites. The fimH SNPs analysis classified the 53 strains into 25 genotype. The genetic fin?gerprintings of 11 isolates were exactly the same. Conclusion Many kinds of virulence factors can be found in ExPEC of el?derly nosocomial infection. The ExPEC strain isolated from non-urine origin had a stronger pathogenicity than those from urine-origin specimens. fimH SNPs analysis is suitable for molecular epidemiological investigation of ExPEC in hospital.

OBJECTIVE ToexplorethepossiblemechanismoractiontargetsofT-2toxinembryo toxicity by observing the effect of T-2 toxin on mitochondrial function of differentiated murine e mbryonic stemcells(mESCs).METHODS Duringdifferentiationat24,72and120h,ESCswereexposedto T-2 toxin 0.5 μg·L-1 .Meanwhile,mESCs were pre-treated with antioxidant Trolox (200 μmol·L-1 )for 30 min and exposed to T-2 toxin (0.5 μg·L-1 )for 72 h.The mitochondrial ultrasture of differentiated mESCs was observed under a transi mission electrical microscope (TEM).The differentiated ESC mito-chondrial function,including respiratory control ratio (RCR),ATP synthase activity and mitochondrial membranepotential(MMP),wasmeasuredat144hafterdifferentiation.RESULTS Significant decrease of the mitochondrial number,deformation of mitochondrial structure,and lack of complete mito-chodrial crest were observed through TEM in the groups of T-2 toxin exposed for 72 and 1 20 h,respec-tively.Compared with the normal control group,RCR declined by 49.5% and 55.1%,ATP synthase activity decreased by 84.9% and 89.3%,and MMP decreased by 23.2% and 35.2% in T-2 toxin 0.5 μg·L-1 exposure 72 and 1 20 h group,respectively.However,the inhibition of mitochondrial function by T-2 toxin in differentiated mESCs recovered significantly in the presence of the antioxidant Trolox. CONCLUSION T-2toxininducesoxidativestressandinhibitsmESCsmitochondrialfunctionindifferenti-ated mESCs,and ROS-induced mitochondrial malfunction plays an i mportant role in T-2 toxin e mbryonic toxicity mechanis m.