Objective:To establish an animal model of trans-sutural distraction osteogenesis in SD rats.Methods:A self-designed V-shaped distraction device(distractor)was fabricated with the traction force(N)of 0,1.3,2.2,3.0,4.3 and 5.0 corresponding to the distraction length(mm)of 5,4,3,2,1 and 0 respectively,meeting the trans-sutural distraction osteogenesis requirements in skull of 5-week-old SD rats.The distractor was plased into the sagittal suture of 12 SD rats.Continuous sampling was conducted 1,3,5 and 7 days respectively(n=3)after operation.The tissue changes in the trans-sutural distraction area were observed by HE and Masson's trichrome staining.Inflammation levels were determined using Arg-1 immunofluorescence staining.The early angiogenesis was clarified through co-staining with CD31 and EMCN.Results:A stable trans-sutural distraction osteogenesis model was estab-lished,5 mm distraction osteogenesis width was observed completely within 7 days of distraction.Significant new bone formation was observed at 7 days after operation.Arg-1 expression increased and was concentrated at the bone margins,overlapping with the areas of new bone formation.EMCN expression gradually decreased,and by day 7 CD31 was predominant,indicating the basic maturation of blood vessels.Conclusion:This study successfully constructed a stable and effective trans-sutural distraction osteogenesis animal model,and provides an experimental basis for the investigation of its early continuous histological changes.

Prostate cancer （PCa） is one of the most common malignant tumors in the male genitourinary system. The phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway is a carcinogenic pathway responsible for the migration， proliferation， and drug resistance of various cancers. In recent years， as the research on the pathogenesis of PCa is deepening， the role of the PI3K/Akt signaling pathway in the development of PCa has attracted much attention. Traditional Chinese medicine， comprehensively regulating multiple components， targets， and pathways， has shown great potential in the treatment of PCa. This article reviews the research progress of traditional Chinese medicine targeting the PI3K/Akt signaling pathway in the treatment of PCa and discusses the expression of the PI3K/Akt signaling pathway in PCa， which involves inhibiting apoptosis of PCa cells， promoting the cell cycle， invasion， and migration of PCa cells， promoting tumor tissue angiogenesis， and mediating the androgen receptor. Additionally， it summarizes the single Chinese medicines that target and regulate this pathway， including Hedyotis diffusa， Taxus chinensis， Bovisc Alculus， and Atractylodis Macrocephalae Rhizoma. The active ingredients of these Chinese medicines mainly include flavonoids， alkaloids， terpenes， polyphenols， lignans， and other compounds. The Chinese medicine compound prescriptions targeting the PI3K/Akt pathway mainly include Wenshen Sanjie prescription， Jianspi Lishi Huayu prescription， Yishen Tonglongtang， Qilan prescription， Xihuangwan， and modified Shenqi Dihuangtang. This review is expected to provide a scientific basis for deeply understanding the pathogenesis of PCa and identifying potential therapeutic targets， as well as to provide new ideas for clinical research and drug development for PCa.

Diabetes mellitus erectile dysfunction(DMED)is a common diabetic-related vascular,endo-crine and neuropathy in clinical practice,and patients with DMED often present with symptoms such as difficulty in erection,prolonged erection time,poor hardness,and short sexual intercourse.The etiological mechanism is complex,and it is often closely related to many factors such as oxidative stress(OS),inflammatory response,and neurological and endocrine lesions,which often cross-react and promote the progression of DMED lesions.In recent years,relevant studies have shown that OS and ferroptosis play a key role in DMED:OS can cause neuro-logical and Abnormal endocrine function,decreased synthesis or bioavailability of penile vascular endothelium,spongy endothelial cell dysfunction and decreased smooth muscle diastolic function,resulting in penile erectile dysfunction,and ferroptosis has also been confirmed to be closely related to DMED,controlling OS and ferroptosis to improve erectile function in diabetic patients is a reasonable and effective treatment pathway,but the mechanism of action of ferroptosis leading to DMED needs to be further studied.Therefore,this article reviews the latest infor-mation on the correlation between OS and ferroptosis and DMED,aiming to provide a useful reference for exploring the mechanism of DMED,clinical prevention and treatment of DMED,and providing potential directions for future research in this field.

Objective To express and purify the glycoprotein extracellular domain (Ex-GP) of Rabies virus strain CTN in soluble form with high efficiency.Methods A recombinant expression plasmid containing the gene encoding the Ex-GP was constructed.Various expression conditions were screened to obtain an optimum prokaryotic expression system for Ex-GP in soluble form.The expressed target protein was purified using affinity chromatography and gel filtration chromatography.Results The target protein Ex-GP with high antigenicity was efficiently expressed in soluble form by using the recombinant PBCX expression system and effectively purified by using affinity and gel filtration chromatography.Conclusion The soluble form of Ex-GP is successfully expressed and purified in a simple and convenient way.This study paves the way for further researches on the biological functions of rabies virus glycoprotein,the pathogenic mechanism of rabies and the development of diagnostic reagent and vaccines for rabies virus.

Objective To investigate the effects of the erythropoietin (EPO) on ischemia reperfusion injury (IRI) in rats with nephron-sparing surgery (NSS). Methods Fifty-four Sprague Dawley rats were divided into 3 groups randomly after right kidney nephrectomy: Sham group, NSS group (PBS+NSS) and EPO group (EPO+NSS). During NSS, renal artery was clamped for 40 min to induce IRI. Sham group just adopted exposure renal artery without vascular clamped. Rats in NSS group were injected intraperitoneally with PBS for 3 days before NSS. Rats in EPO group were injected intraperitoneally with EPO for 3 days before NSS. After 12 h, 24 h, 72 h, blood sample and renal tissues were collected. The serum creatinine (Scr) and urea nitrogen (BUN) were evaluated. The pathology injury was evaluated by HE staining. The CD24/CD133 double-positived renal progenitor cells (RPCs) were tested by flow cytometry. The CD133 and PCNA protein were quantified by immunohistochemical staining. The expressions of Wnt7b and β-catenin protein were detected by Western blotting. Results Rats in NSS group had more elevated Scr, BUN and pathology injury scores 12 h, 24 h and 72 h after operation than those in Sham group (all P<0.05). Compared with those in the NSS group, the Scr and BUN in the EPO group were significantly lower 24 h after the surgery (all P<0.05), and the pathology injury score also decreased (P<0.05). The proportion of RPCs, expressions of CD133 and PCNA, and expressions of Wnt7b and β-catenin protein were significantly higher after 24 h of the surgery in NSS group than those in the Sham group (all P<0.05). While compared with those in the NSS group, the proportion of RPCs and expressions of CD133, PCNA, Wnt7b and β-catenin increased at the EPO group (all P<0.05). Conclusions EPO can reduce the IRI after NSS, and its mechanism may be related to the mobilization of the RPCs by the Wnt7b/β-catenin signal pathway.

Objective To prepare a neutralizing monoclonal antibody against rabies virus. Meth-ods BALB/c mice were immunized with the inactivated rabies virus CTN strains on day 0, 7, 14 and 28. Spleen cell samples were collected and then fused with SP2/0 cells to prepare the hybridoma cell line. Posi-tive hybridoma cells that were screened out with RFFIT technique were injected into BALB/c mice intraper-itoneally. Ascites samples were collected from the mice to separate neutralizing monoclonal antibodies. Affin-ity chromatography was used for the purification of neutralizing monoclonal antibodies. Subtype identification and sequencing analysis were performed for further identification. A colloidal gold strip based method for rap-id detection of rabies vaccine was established with the prepared monoclonal antibodies. Results The hybri-doma cell line, CTN-McAb1, was prepared successfully with stable secretion of neutralizing monoclonal anti-bodies against rabies virus. The purity of those antibodies was more than 95% after purification and the sub-type of them was IgG1. The colloidal gold strip for raid detection of rabies vaccine was successful prepared. Conclusion The neutralizing monoclonal antibody against rabies virus was successfully prepared and could be used for preliminary application. This study will be of great significance for the quality control of rabies vaccine.

Objective:To investigate whether endogenous hydrogen sulfide (H2 S)was involved in the pathogenesis of osteoarthritis (OA)and its underlying mechanism,to detect H2 S and its synthases ex-pression in knee cartilage in patients diagnosed with different severity of OA,and to explore the transcrip-tion and expression of gene MMP-13 in chondrocytes treated with IL-1βor H2S.Methods:Synovial fluids of the in-patients with different severity of OA hospitalized in Peking University First Hospital were collected for measurement of H2 S content using methylene blue assay.Articular cartilages of the patients who underwent knee arthroplasty were collected for the cell culture of relatively normal chondrocytes.The chondrocytes were cultured to the P3 generation and H2 S molecular probes were used for detection of endogenous H2 S generation in the chondrocytes.Immunocytochemistry was used to detect the localization of H2 S synthases including cystathionine β-synthase (CBS),cystathionine-γ-lyase (CSE),and mercap-topyruvate sulfurtransferase (MPST)in OA chondrocytes.Western blot was used to quantify the protein expressions of CSE,MPST,and CBS in cartilage tissues of the patients who were diagnosed with OA and underwent knee arthroplasty.The relatively normal human chondrocytes were cultured to passage 3 and then divided into 4 groups for different treatments:(1 )the normal control group,no reagent was added;(2)the IL-1βgroup,5 μg/L of IL-1βwas added;(3)the IL-1β+H2S group,200 μmol/L of NaHS was added 30 min before adding 5 μg/L of IL-1β;(4)the H2 S group,200 μmol/L of NaHS was added. The transcription and expression of gene MMP-13 in chondrocytes of each group were determined with Real-time PCR and Western blot,respectively.And the total NF-κB p65 and phosphorylated NF-κB p65 in chondrocytes were detected with Western blot.Results:The content of H2 S in the synovial fluid of degenerative knee was (14.3 ±3.3)μmol/L.Expressions of endogenous H2 S and its synthases including CBS,CSE and MPST were present in the cytoplasm of chondrocytes.CSE protein expression in Grade 3 (defined by outerbridge grading)cartilage tissues was significantly increased as compared with that of Grade 1 cartilage tissues (1.67 ±0.09 vs.1.26 ±0.11,P<0.05).However,no significant difference of CBS or MPST expression among the different groups was observed.The expression of MMP-13 protein in the IL-1βgroup was significantly higher than that in the normal chondrocytes (1 .87 ±0.67 vs.0.22 ± 0.10,P<0.05 ),and that in the IL-1β+H2 S group was significantly decreased than that in the IL-1βgroup (0.55 ±0.11 vs.1.87 ±0.67,P<0.05),and that in the H2S group had no significant difference compared with that in the normal control group.The transcription of MMP-13 protein in the IL-1βgroup was significantly higher than that in the normal chondrocytes (31.40 ±0.31 vs.1.00 ±0.00,P<0.05), and that in the IL-1β+H2 S group was significantly decreased than that in the IL-1βgroup (24.41 ± 1.28 vs.31.40 ±0.31,P<0.05),and that in the H2S group had no significant difference compared with that in the normal control group.The total NF-κB p65 in the IL-1βgroup was significantly higher than that in the normal chondrocytes (2.13 ±0.08 vs.0.73 ±0.08,P<0.05),and that in the IL-1β+H2S group was significantly decreased than that in the IL-1βgroup (1 .24 ±0.13 vs.2.13 ±0.08,P<0.05 ),and that in the H2 S group had no significant difference compared with that in the normal control group.The phosphorylated NF-κB p65 in IL-1βgroup was significantly higher than that in the normal chondrocytes (1.30 ±0.13 vs.0.19 ±0.04,P<0.05),and that in IL-1β+H2S group was significantly decreased than that in the IL-1βgroup (0.92 ±0.26 vs.1.30 ±0.13,P<0.05),and that in the H2S group had no significant difference compared with that in the normal control group.Conclusion:H2 S affected the cartilage degeneration by partly inhibiting the degradation of extracellular matrix.

Objective To study to take the oxidized Mannan?modified 786?0 in renal clear cell carcinom as tumor cells antigen to sensitize Dendrit?ic cells(DC)and to observe the its killing effect on renal clear cell carcinoma of CTLs induced. Methods Getting the peripheral blood mononucle?ar cells from the volunteers,and then to be stimulated to turn to be maturation by GM?CSF and IL?4 in vitro. Taking the clear renal carcinoma cell as the tumor antigen,and then making it to be modified by oxidized Mannan to acquire the tumor cell vaccine.Experimental groups include:blank group:DC?PBS group,control group:control?DC?786?0,experimental group:DC?Ox?Mannose?786?0 group. Taking the flow cytometry to detect the changes of DC phenotype,then taking the ELISA to detect the sencretion levels of supernatant of IL?12 of DC,then taking the CCK to detecte the cytotoxicity of lymphocytes(CTL)induced by DC of these experiment groups. Results Results by flow cytometry:the mature phenotype of DCs sensitized by Ox?Mannose?786?0 group included CD80,CD83,CD86 and HLA?DR expressed significantly higher than the other groups. As well as the secretion levels of IL?12. Meanwhile the cytotoxicity activity of lymphocytes(CTLs)induced by DCs which are sensitized by Ox?Mannose?786?0 increased more significantly than the other groups. Conclusion Glycosylated Antigens can be more effective in sensitizing antigen?presenting cells DC,and stimulating them to be maturation,while the killing effect to tumor cells also have noticeably improved.

Objective To construct the eukaryotic expression plasmid of the rabies virus glycoprotein gene DNA,and detect the immunogenicity.Methods Using RT-PCR amplified the glycoprotein gene of rabies virus CTN strain,sequenced and cloned into pcDNA5.0 (+) vector to construct the recombinant plasmid pcDNA5.0-G plasmid.Detect glycoprotein transient expression with transfecting the plasmid into 293T cells.Intramuscular immunization of BALB/c mice by the recombinant plasmid on day 0 and 7,then challenge by rabies virus CVS strain observed the mice survived.Results The results of the transient expression of glycoprotein abundantly expressed.The survival ratio of mice with CVS challenge after routine intramuscular injection of pcDNA5.0-G plasmid is 73.3％,and 6.7％ for the control group.Conclusion Rabies virus glycoprotein eukaryotic expression plasmid pcDNA5.0-G was successfully constructed,and has been good immunogenicity.It's to be the foundation for candidate DNA vaccine research and development.