Identification of an Epoxide Metabolite of Amitriptyline In Vitro and In Vivo.

Amitriptyline (ATL), a tricyclic antidepressant, has been reported to cause various adverse effects, particularly hepatotoxicity. The mechanisms of ATL-induced hepatotoxicity remain unknown. The study was performed to identify the olefin epoxidation metabolite of ATL and determine the possible toxicity mechanism. Two glutathione (GSH) conjugates (M1 and M2) and two N-acetylcysteine (NAC) conjugates (M3 and M4) were detected in rat liver microsomal incubations supplemented with GSH and NAC, respectively. Moreover, M1/M2 and M3/M4 were respectively found in ATL-treated rat primary hepatocytes and in bile and urine of rats given ATL. Recombinant P450 enzyme incubations demonstrated that CYP3A4 was the primary enzyme involved in the olefin epoxidation of ATL. Treatment of hepatocytes with ATL resulted in significant cell death. Inhibition of CYP3A attenuated the susceptibility to the observed cytotoxicity of ATL. The metabolic activation of ATL most likely participates in the cytotoxicity of ATL.

Esophageal cancer-related gene 4 inhibits gastric cancer growth and metastasis by upregulating Krüppel-like factor 2 expression.

Background: There are a large number of people suffering from gastric cancer (GC) worldwide, so the study of biomarkers for GC is urgently needed. This study aimed to investigate the role of esophageal cancer-related gene 4 (ECRG4) in the growth, metastasis, and prognosis of GC and the possible underlying mechanism. Methods: The expression of ECRG4 was detected in GC tissues by quantitative polymerase chain reaction (PCR), Western blot, and immunohistochemistry. The relationships between ECRG4 expression and clinicopathological parameters of patients with GC were statistically analyzed, and Kaplan-Meier prognosis and survival curves of the patients were plotted. ECRG4 was overexpressed in the human gastric adenocarcinoma cell line (AGS) and human GC cell line 27 (HGC27), and the in vivo effects of ECRG4 overexpression on the growth, invasion, and metastasis of GC were analyzed and verified in nude mice. To identify the downstream transcription factors potentially regulated by ECRG4, ribonucleic acid (RNA) sequencing and differential gene expression analysis were performed on ECRG4-overexpressing cells. Quantitative PCR, Western blot, and immunohistochemistry were used to detect the expression of the downstream transcription factors targeted by ECRG4 in GC. Results: The ECRG4 mRNA and protein expression levels were low in GC tissues and were associated with a poor prognosis. Least absolute shrinkage and selection operator (LASSO) Cox regression and Kaplan-Meier survival analyses showed that patients with low ECRG4 expression had worse prognosis and survival. Overexpression of ECRG4 inhibited the proliferation, metastasis, and invasion of GC cells. RNA sequencing analysis showed that overexpression of ECRG4 induced the upregulation of Krüppel-like factor 2. Conclusions: Our findings show that ECRG4 promotes GC progression via Krüppel-like factor 2 signaling and highlight ECRG4 as a potential GC biomarker and therapeutic target.

CYP2C19 gene polymorphism in Ningxia.

BACKGROUND: Poor metabolizer (PM) status of CYP2C19 can be a predisposing factor for developing gastric cancer in H. pylori-infected patients. It is unclear whether PM status of CYP2C19 can also be a potential factor for H.pylori infection in healthy people. METHODS: We used high-throughput sequencing to detect single nucleotide polymorphisms (SNPs) at just three loci, rs4244285 (CYP2C19*2), rs4986893 (CYP2C19*3) and rs12248560 (CYP2C19*17), to identify the exact CYP2C19 alleles corresponding to the mutated sites. We determined CYP2C19 genotypes of 1050 subjects from 5 cities of Ningxia from September 2019 to September 2020 and evaluated the potential correlation between H.pylori and CYP2C19 gene polymorphisms. Clinical data were analyzed using χ2 tests. RESULTS: The frequency of CYP2C19*17 in Hui (3.7%) was higher as compared to Han (1.4%) in Ningxia (p = 0.001). The frequency of CYP2C19*1/*17 of Hui (4.7%) was higher as compared to Han (1.6%) in Ningxia (p = 0.004). The frequency of CYP2C19*3/*17 of Hui (1%) was higher as compared to Han (0%) in Ningxia (p = 0.023). The frequencies of alleles (p = 0.142) and genotypes (p = 0.928) were not found to be significantly different among the different BMI groups. The frequencies of four alleles between H. pylori positive and negative groups were not found to be statistically different (p = 0.794). The frequencies of the different genotypes between H. pylori positive and negative groups were not statistically different (p = 0.974), and no statistical difference was observed between the different metabolic phenotypes (p = 0.494). CONCLUSION: There were regional differences observed in CYP2C19*17 distribution in Ningxia. The frequency of CYP2C19*17 in Hui was higher than in Han of Ningxia. No significant relationship was found between CYP2C19 gene polymorphism and susceptibility to H. pylori infection.

Krüppel-Like Factor 2 Is a Gastric Cancer Suppressor and Prognostic Biomarker.

Gastric cancer (GC) is a common digestive tract tumor. Due to its complex pathogenesis, current diagnostic and therapeutic effects remain unsatisfactory. Studies have shown that KLF2, as a tumor suppressor, is downregulated in many human cancers, but its relationship and role with GC remain unclear. In the present study, KLF2 mRNA levels were significantly lower in GC compared to adjacent normal tissues, as analyzed by bioinformatics and RT-qPCR, and correlated with gene mutations. Tissue microarrays combined with immunohistochemical techniques showed downregulation of KLF2 protein expression in GC tissue, which was negatively correlated with patient age, T stage, and overall survival. Further functional experiments showed that knockdown of KLF2 significantly promoted the growth, proliferation, migration, and invasion of HGC-27 and AGS GC cells. In conclusion, low KLF2 expression in GC is associated with poor patient prognosis and contributes to the malignant biological behavior of GC cells. Therefore, KLF2 may serve as a prognostic biomarker and therapeutic target in GC.

Nomogram for Postoperative Headache in Adult Patients Undergoing Elective Cardiac Surgery.

Background Postoperative headache (POH) is frequent after cardiac surgery; however, few studies on risk factors for POH exist. The aims of the current study were to explore risk factors related to POH after elective cardiac surgery and to establish a predictive system. Methods and Results Adult patients undergoing elective open-heart surgery under cardiopulmonary bypass from 2016 to 2020 in 4 cardiac centers were retrospectively included. Two thirds of the patients were randomly allocated to a training set and one third to a validation set. Predictors for POH were selected by univariate and multivariate analysis. POH developed in 3154 of the 13 440 included patients (23.5%) and the overall mortality rate was 2.3%. Eight independent risk factors for POH after elective cardiac surgery were identified, including female sex, younger age, smoking history, chronic headache history, hypertension, lower left ventricular ejection fraction, longer cardiopulmonary bypass time, and more intraoperative transfusion of red blood cells. A nomogram based on the multivariate model was constructed, with reasonable calibration and discrimination, and was well validated. Decision curve analysis revealed good clinical utility. Finally, 3 risk intervals were divided to better facilitate clinical application. Conclusions A nomogram model for POH after elective cardiac surgery was developed and validated using 8 predictors, which may have potential application value in clinical risk assessment, decision-making, and individualized treatment associated with POH.

Nomogram Models to Predict Postoperative Hyperlactatemia in Patients Undergoing Elective Cardiac Surgery.

Objectives: Postoperative hyperlactatemia (POHL) is common in patients undergoing cardiac surgery and is associated with poor outcomes. The purpose of this study was to develop and validate two predictive models for POHL in patients undergoing elective cardiac surgery (ECS). Methods: We conducted a multicenter retrospective study enrolling 13,454 adult patients who underwent ECS. All patients involved in the analysis were randomly assigned to a training set and a validation set. Univariate and multivariate analyses were performed to identify risk factors for POHL in the training cohort. Based on these independent predictors, the nomograms were constructed to predict the probability of POHL and were validated in the validation cohort. Results: A total of 1,430 patients (10.6%) developed POHL after ECS. Age, preoperative left ventricular ejection fraction, renal insufficiency, cardiac surgery history, intraoperative red blood cell transfusion, and cardiopulmonary bypass time were independent predictors and were used to construct a full nomogram. The second nomogram was constructed comprising only the preoperative factors. Both models showed good predictive ability, calibration, and clinical utility. According to the predicted probabilities, four risk groups were defined as very low risk (<0.05), low risk (0.05-0.1), medium risk (0.1-0.3), and high risk groups (>0.3), corresponding to scores of ≤ 180 points, 181-202 points, 203-239 points, and >239 points on the full nomogram, respectively. Conclusions: We developed and validated two nomogram models to predict POHL in patients undergoing ECS. The nomograms may have clinical utility in risk estimation, risk stratification, and targeted interventions.

Development and Validation of Nomogram Models for Postoperative Pneumonia in Adult Patients Undergoing Elective Cardiac Surgery.

Background: Postoperative pneumonia (POP) is a frequent complication following cardiac surgery, related to increased morbidity, mortality and healthcare costs. The objectives of this study were to investigate the risk factors associated with POP in adults undergoing elective cardiac surgery and to develop and validate nomogram models. Methods: We conducted a multicenter retrospective study in four cardiac centers in China. Adults operated with elective open-heart surgery from 2016 to 2020 were included. Patients were randomly allocated to training and validation sets by 7:3 ratio. Demographics, comorbidities, laboratory data, surgical factors, and postoperative outcomes were collected and analyzed. Risk factors for POP were identified by univariate and multivariate analysis. Nomograms were constructed based on the multivariate logistic regression models and were evaluated with calibration, discrimination and decision curve analysis. Results: A total of 13,380 patients meeting the criteria were included and POP developed in 882 patients (6.6%). The mortality was 2.0%, but it increased significantly in patients with POP (25.1 vs. 0.4%, P < 0.001). Using preoperative and intraoperative variables, we constructed a full nomogram model based on ten independent risk factors and a preoperative nomogram model based on eight preoperative factors. Both nomograms demonstrated good calibration, discrimination, and were well validated. The decision curves indicated significant clinical usefulness. Finally, four risk intervals were defined for better clinical application. Conclusions: We developed and validated two nomogram models for POP following elective cardiac surgery using preoperative and intraoperative factors, which may be helpful for individualized risk evaluation and prevention decisions.

Incidence, Risk Factors and Outcomes of Postoperative Headache After Stanford Type a Acute Aortic Dissection Surgery.

Background: Postoperative headache (POH) is common in clinical practice, however, no studies about POH after Stanford type A acute aortic dissection surgery (AADS) exist. This study aims to describe the incidence, risk factors and outcomes of POH after AADS, and to construct two prediction models. Methods: Adults who underwent AADS from 2016 to 2020 in four tertiary hospitals were enrolled. Training and validation sets were randomly assigned according to a 7:3 ratio. Risk factors were identified by univariate and multivariate logistic regression analysis. Nomograms were constructed and validated on the basis of independent predictors. Results: POH developed in 380 of the 1,476 included patients (25.7%). Poorer outcomes were observed in patients with POH. Eight independent predictors for POH after AADS were identified when both preoperative and intraoperative variables were analyzed, including younger age, female sex, smoking history, chronic headache history, cerebrovascular disease, use of deep hypothermic circulatory arrest, more blood transfusion, and longer cardiopulmonary bypass time. White blood cell and platelet count were also identified as significant predictors when intraoperative variables were excluded from the multivariate analysis. A full nomogram and a preoperative nomogram were constructed based on these independent predictors, both demonstrating good discrimination, calibration, clinical usefulness, and were well validated. Risk stratification was performed and three risk intervals were defined based on the full nomogram and clinical practice. Conclusions: POH was common after AADS, portending poorer outcomes. Two nomograms predicting POH were developed and validated, which may have clinical utility in risk evaluation, early prevention, and doctor-patient communication.

Orexin A associates with inflammation by interacting with OX1R/OX2R receptor and activating prepro-Orexin in cancer tissues of gastric cancer patients.

OBJECTIVE: Gastric cancer (GC) has been become the second leading cause for cancer-associated death. This study aimed to investigate Orexin A levels and associated receptors in tumor tissues of GC patients. PATIENTS AND METHODS: Forty-six consecutive gastric cancer patients (GC, n=46) and 13 chronic atrophic gastritis patients (CAG, n=13) were recruited. Meanwhile, 18 health individuals visiting Medical Examination Department were involved as control (N group, n=18). ELISA was used to examine Orexin A concentration. Immunohistochemistry assay was used to examine OX1R and OX2R. HE staining was applied to evaluate inflammation. qRT-PCR was employed to detect OX1R, OX2R, prepro-Orexin mRNAs. Serum Helicobacter pylori (H. pylori) infection was measured. RESULTS: Orexin A expression in GC patients was significantly up-regulated compared to N group and CAG group (p<0.05). Orexin A expression was increased in CAG group compared to N group (p<0.05). Gastric cancer tissues exhibited significantly obvious inflammation compared to N group and CAG group (p<0.05). OX1R and OX2R expressions were significantly down-regulated in GC group compared to N group and CAG group (p<0.05). OX1R and OX2R were lower significantly in GC group compared to CAG group (p<0.05). Prepro-Orexin was significantly depleted in tumor tissues of GC group compared to N group and CAG group (p<0.05). Orexin A expression was un-associated with gender, age and differential grades (p>0.05). CAG and GC patients demonstrated higher H. pylori infection rates. CONCLUSION: Orexin A was associated with inflammation by interacting with OX1R/OX2R receptor and activating prepro-Orexin in tumor tissues of gastric cancer patients.

The Therapeutic Effect of ICAM-1-Overexpressing Mesenchymal Stem Cells on Acute Graft-Versus-Host Disease.

BACKGROUND/AIMS: Mesenchymal stem cells (MSCs) do not readily migrate to appropriate sites, and this creates a major obstacle for their use in the treatment of graft-versus-host disease (GVHD). Intercellular adhesion molecule-1 (ICAM-1) can guide the homing of various immune cells to the proper anatomical location within secondary lymphoid organs (SLOs), which are the major niches for generating immune responses or tolerance. MSCs rarely migrate to SLOs after intravenous infusion, and are constitutively low expression of ICAM-1. So in our previous work, ICAM-1 was engineered into a murine MSC line C3H10T1/2 by retrovirus transfection system (ICAM-1MSCs). Here, we hypothesized that ICAM-1highMSCs may significantly improve their immunomodulatory effect. METHODS: We used different co-culture methods combined with real-time PCR and flow cytometry to evaluate ICAM-1highMSCs immunomodulatory effect on dendritic cells (DCs) and T cells in vitro and in vivo. MSCs were labeled with carboxyfluorescein diacetate succinimidylester (CFSE) to detect its distribution in mouse model. RESULTS: Our in vitro analyses revealed ICAM-1 MSCs could suppress DCs maturation according to co-culture methods and suppress the T cell immune response according to the mixed lymphocyte response (MLR) and lymphoblast transformation test (LTT) tests. We found that infusion of ICAM-1highMSCs potently prolonged the survival of GVHD mouse model. The infused ICAM-1highMSCs migrate to SLOs in vivo, and suppressed DCs maturation, suppressed CD4+ T cell differentiation to Th1 cells, and increased the ratios of Treg cells. CONCLUSIONS: Taken together, these data demonstrate that ICAM-1highMSCs had an enhanced immunosuppressive effect on DCs and T cells, which may help explain the protective effect in a GVHD model. This exciting therapeutic strategy may improve the clinical efficacy of MSC-based therapy for GVHD.

Downregulated CDK10 expression in gastric cancer: Association with tumor progression and poor prognosis.

The tumor suppressor characteristics of cyclin­dependent kinase 10 (CDK10) in nasopharyngeal carcinoma and breast cancer have been previously demonstrated. In the present study the expression status of CDK10 and its prognostic significance in gastric cancer was determined, as well as its role in cell proliferation and invasion. Immunoblot analysis revealed that CDK10 protein expression was notably decreased in gastric cancer compared with normal tissues. Immunohistochemistry demonstrated that the loss of CDK10 expression, which was observed in 50.8% of primary gastric cancer tissues (n=128), significantly correlated with advanced tumor stage (P<0.001), frequent lymph node metastasis (P<0.001), distant metastasis (P=0.013), tumor differentiation (P=0.004) and unfavorable overall survival (P<0.001). Multivariate analysis suggested that CDK10 expression may serve as an independent prognostic predictor (P=0.001) for the progression of gastric cancer. In addition, ectopic CDK10 expression inhibited gastric cancer cell proliferation, migration and invasion, while knockdown of CDK10 promoted these phenotypes. Collectively, the results of the present study indicated that CDK10 expression may serve as a novel prognostic biomarker that holds therapeutic promise for gastric cancer.

Structure, evolution, and comparative genomics of tetraploid cotton based on a high-density genetic linkage map.

A high-density linkage map was constructed using 1,885 newly obtained loci and 3,747 previously published loci, which included 5,152 loci with 4696.03 cM in total length and 0.91 cM in mean distance. Homology analysis in the cotton genome further confirmed the 13 expected homologous chromosome pairs and revealed an obvious inversion on Chr10 or Chr20 and repeated inversions on Chr07 or Chr16. In addition, two reciprocal translocations between Chr02 and Chr03 and between Chr04 and Chr05 were confirmed. Comparative genomics between the tetraploid cotton and the diploid cottons showed that no major structural changes exist between DT and D chromosomes but rather between AT and A chromosomes. Blast analysis between the tetraploid cotton genome and the mixed genome of two diploid cottons showed that most AD chromosomes, regardless of whether it is from the AT or DT genome, preferentially matched with the corresponding homologous chromosome in the diploid A genome, and then the corresponding homologous chromosome in the diploid D genome, indicating that the diploid D genome underwent converted evolution by the diploid A genome to form the DT genome during polyploidization. In addition, the results reflected that a series of chromosomal translocations occurred among Chr01/Chr15, Chr02/Chr14, Chr03/Chr17, Chr04/Chr22, and Chr05/Chr19.

Subhaplogroup D4b1 enhances the risk of cervical cancer initiation: A case-control study in southern China.

AIM: To investigate whether mitochondrial DNA (mtDNA) background (haplogroup) is associated with cervical cancer in patients in southern China. METHODS: A case-control study of 150 patients with cervical cancer and 217 geographically matched controls was conducted in Wenzhou, a southern Chinese city in the Zhejiang province. DNA from peripheral blood was extracted and sequenced. Sequences were aligned to the mtDNA revised Cambridge Reference Sequence (GenBank number NC_012920) to determine mtDNA single nucleotide polymorphisms (SNPs) and haplogroups. RESULTS: We found that both M and N haplogroups and their diagnostic SNPs (A10398G and C10400T) are not associated with the risk of cervical cancer. However, individuals with haplogroup D4b1/D4b1*, an M subhaplogroup, exhibited an increased risk of cervical cancer (odds ratio [OR] = 1.034; 95% confidence interval [CI] 1.004, 1.066; P = 0.011/OR =1.027; 95% CI 1.001, 1.055; P = 0.027). Individuals with SNPs C10181T/A10136G (OR =1.034; 95% CI 1.004, 1.066; P = 0.011/OR =1.027; 95% CI 1.001, 1.055; P = 0.027) were more susceptible to cervical cancer than individuals without. Furthermore, we determined that mtDNA background is not associated with the progression of cervical cancer. CONCLUSIONS: Our results indicate that mtDNA haplogroups play a role in cervical cancer initiation.

Mesenchymal stem cells attenuated PLGA-induced inflammatory responses by inhibiting host DC maturation and function.

The poly lactic-co-glycolic acid (PLGA) bio-scaffold is a biodegradable scaffold commonly used for tissue repair. However, implanted PLGA scaffolds usually cause serious inflammatory responses around grafts. To improve PLGA scaffold-based tissue repair, it is important to control the PLGA-mediated inflammatory responses. Recent evidence indicated that PLGA induce dendritic cell (DC) maturation in vitro, which may initiate host immune responses. In the present study, we explored the modulatory effects of mesenchymal stem cells (MSC) on PLGA-induced DCs (PLGA-DC). We found that mouse MSCs inhibited PLGA-DC dendrite formation, as well as co-stimulatory molecule and pro-inflammatory factor expression. Functionally, MSC-educated PLGA-DCs promoted Th2 and regulatory T cell differentiation but suppressed Th1 and Th17 cell differentiation. Mechanistically, we determined that PLGA elicited DC maturation via inducing phosphorylation of p38/MAPK and ERK/MAPK pathway proteins in DCs. Moreover, MSCs suppressed PLGA-DCs by partially inactivating those pathways. Most importantly, we found that the MSCs were capable of suppressing DC maturation and immune function in vivo. Also, the proportion of mature DCs in the mice that received MSC-PLGA constructs greatly decreased compared with that of their PLGA-film implantation counterparts. Additionally, MSCs co-delivery increased regulatory T and Th2 cells but decreased the Th1 and Th17 cell numbers in the host spleens. Histological analysis showed that MSCs alleviated the inflammatory responses around the grafted PLGA scaffolds. In summary, our findings reveal a novel function for MSCs in suppressing PLGA-induced host inflammatory response and suggest that DCs are a new cellular target in improving PLGA scaffold-based tissue repair.

Immune-enhancing effect of nano-DNA vaccine encoding a gene of the prME protein of Japanese encephalitis virus and BALB/c mouse granulocyte-macrophage colony-stimulating factor.

Plasmid-encoded granulocyte-macrophage colony-stimulating factor (GM­CSF) is an adjuvant for genetic vaccines; however, how GM-CSF enhances immunogenicity remains to be elucidated. In the present study, it was demonstrated that injection of a plasmid encoding the premembrane (prM) and envelope (E) protein of Japanese encephalitis virus and mouse GM-CSF (pJME/GM-CSF) into mouse muscle recruited large and multifocal conglomerates of macrophages and granulocytes, predominantly neutrophils. During the peak of the infiltration, an appreciable number of immature dendritic cells (DCs) appeared, although no T and B-cells was detected. pJME/GM-CSF increased the number of splenic DCs and the expression of major histocompatibility complex class II (MHCII) on splenic DC, and enhanced the antigenic capture, processing and presentation functions of splenic DCs, and the cell-mediated immunity induced by the vaccine. These findings suggested that the immune-enhancing effect by pJME/GM-CSF was associated with infiltrate size and the appearance of integrin αx (CD11c)+cells. Chitosan-pJME/GM-CSF nanoparticles, prepared by coacervation via intramuscular injection, outperformed standard pJME/GM-CSF administrations in DC recruitment, antigen processing and presentation, and vaccine enhancement. This revealed that muscular injection of chitosan­pJME/GM-CSF nanoparticles may enhance the immunoadjuvant properties of GM-CSF.

[Influence of donor mouse age on the establishment of murine acute graft versus host disease model after allogenic hematopoietic stem cell transplantation].

This study was purposed to elucidate the influence of donor mouse age on the establishment of murine acute graft versus host disease (aGVHD) model after allogenic hematopoietic stem cell transplantation. The male mice with 2-week-old, 10-week-old and 18-week-old mice (BALB/cH-2Kb) were taken as donors. The 8-week-old mice (BALB/c, H-2Kd) were selected as recipients. Each group animals were irradiated with 7.5 Gy (60)Co for total body, the recipient mice were injected intravenously with 1 × 107 bone marrow cells and 1 × 107 spleenoctyes from various donors in 4-5 hours after irradiation. Mouse transplant characteristics and survival were observed every day. The white blood cell number in peripheral blood of each group were counted at day 5, 10, 15, 20, 25 and 30 after transplantation. Furthermore, the pathological damage in the liver, spleen, lung and intestines were evaluated by sectioning and in situ hematoxylin-eosin (HE) staining. The results showed that compared with the 2-week-old and 10-week-old donor groups, mice received bone marrow (BM) cells and splenocytes from 18-week-old mice showed higher incidence of aGVHD, lower clinical GVHD scores and suffered from diarrhea, ruffled hair, a hunched posture, and diminished body weight. In contrast, mice received BM cells and splenocytes from 2-week-old donor mice indicated attenuated GVHD symptoms and survived longer. The histo-pathological analysis in 18-week-old donor group demonstrated the most serious pathological damage in the liver, spleen, lung and intestines. It is concluded that the donor age has been confired to have an obvious influence on the establishment of murine aGVHD model. This study lay an important foundation for establishing animal models and may be helpful for further study.

[Construction of mouse VCAM-1 expression vector and establishment of stably transfected MSC line C3H10T1/2].

This study was aimed to construct the mouse VCAM-1 expression vector, to establish the stably transfected MSC line and to investigate the effect of VCAM-1-modified mesenchymal stem cells (MSC) on the immunological characteristics of MSC. The cDNA of murine VCAM-1 gene was amplified by RT-PCR from the total RNA isolated from the mouse spleen; then the cDNA was inserted into the retrovirus vector PMSCVmigr-1; the recombinant plasmid was confirmed by restriction endonuclease experiments and sequencing, then designated as PMSCVmigr-1-mVCAM-1; the recombinant plasmid PMSCVmigr-1-mVCAM-1 was transfected into 293 cells by lipofecamin and the supernatant was collected to transfect MSC cell line (C3H10T1/2). Moreover, VCAM-1 expression on MSC was evaluated by FACS. Furthermore, the inhibitory effect of VCAM-1-MSC on lymphocytic transformation was tested by (3)H-TdR incorporation assay. The results indicated that the successful construction of recombinant retroviral expression plasmid of mouse VCAM-1 was confirmed by digesting and sequancing. After transfection of MSC with retroviral supernaptant, the high expression of VCAM-1 on MSC could be detected by flow cytometry. The MSC high expressing VCAM-1 could significantly inhibit the proliferation of Con A-inducing lymphocytes in dose-depentent marrer. It is concluded that recombinant retroviral encoding VCAM-1 (PMSCVmigr-1-mVCAM-1) has been successfully constructed and mouse VCAM-1 has been stably expressed in C3H10T1/2. MSC over-expressing VCAM-1 show more potent immunosuppressive effect on cellular immune reaction in vitro. Our data laid a foundation for the subsequent studying the effect of VCAM-1 transfecting into MSC on immune related disease study.

[Effect of different irradiation doses on the establishment of murine cGVHD model after MHC matched spleen stem cell transplantation].

This study was aimed to investigate the effect of different irradiation doses on the establishment of murine cGVHD model after MHC matched spleen stem cell transplantation. The male mouse BALB/c(H)-2d was totally irradiated with different radiation dose of (60)Co (TBI), then was infused with the same number of splenocytes from MHC matched DBA/2 male mice. After transplantation, the bodyweight, general appearance, hair changes, survival time and pathological damage were observed. The results indicated that compared to the control group (0 Gy) and the 7.0 Gy group, the mice irradiated with 7.5 Gy and 8.0 Gy showed cGVHD symptoms and obvious pathological damage. At the end of experiments (60 d after transplantation), all mice irradiated by 7.5 Gy survived while only 60% animals survived in the 8.0 Gy group. It is concluded that under infusion of 10(8) MHC matched splenocytes per mouse, 7.5 Gy irradiation is appropriate to efficiently establish cGVHD model. This study laid an important foundation for further studying the pathogenesis, biological characteristics, and intervention factors of cGVHD.

[Effect of intercellular adhesion molecule-1 on the migration in vitro of murine mesenchymal stem cells and its related mechanism].

This study was aimed to investigate the effect of intercellular adhesion molecule-1 (ICAM-1) on the migration in vitro of the murine mesenchymal stem cells (MSC) and its related mechanisms. The migration ability of murine MSC (C3H10T1/2), ICAM-1 transfected MSC (C3H10T1/2-MIGR1-ICAM-1) and empty vector-transfected MSC (C3H10T 1/2-MIGR1) were assayed in vitro by using the transwell system. Briefly, the cells were seeded on the membrane with 8 µm aperture and the fetal bovine serum was used as the chemotactic agent to induce MSC migration. The transmigrated cells were stained by crystal purple as well as DAPI for 8 h and 12 h respectively. The absolute cell numbers were counted and the migration rates of MSC were evaluated in each group. To explore the potential mechanisms which control the migration of MSC, the specific chemical inhibitors of MAPK pathway (SB203580, PD98059 and JNK inhibitor II) were added to the transwell system and the alteration of the MSC migration ability were evaluated at 12 h. The results showed that the migration ability at 8 h and 12 h of the ICAM-1-transfected MSC increased. Both absolute cell number and migration rate of MSC were significantly up-regulated by ICAM-1. Furthermore, the promoting effect of ICAM-1 on migration was partially suppressed by the inhibition of JNK/SAPK pathway. The transmigrated cell numbers and the migration rate decreased with the addition of JNK inhibitor II. However, the ICAM-1 promoting migration of MSC was not suppressed by the inhibitors for ERK/MAPK and p38/MAPK pathway did not work in the present study. It is concluded that ICAM-1 can induce mouse MSC migration in vitro, and the promoting effect is partially dependent on the activation of JNK/SAPK pathway.

[Latest research progress on pathogenesis of chronic graft versus host disease and its related problems].

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective method for the treating of malignant diseases of hematopoietic system or non-malignant proliferative diseases, but the occurrence of graft-versus-host disease (GVHD) limits the success rate of hematopoietic stem cell transplantation. Moreover, chronic graft-versus-host disease (cGVHD) is the main factor affecting the long-term survival rate and life quality of recipient after hematopoietic stem cell transplantation. In this article, the latest research progress of the pathogenesis of cGVHD and related problems are reviewed from the thymus, cytokines, T lymphocyte subsets, B lymphocytes and its secreted antibody.