Prognostic influencing factors analysis of patients with estrogen receptor-positive de novo stage Ⅳbreast cancer / 肿瘤研究与临床

Objective:To investigate the factors influencing the prognosis of patients with estrogen receptor (ER)-positive de novo stage Ⅳ breast cancer.Methods:The clinical data of 339 patients with ER-positive de novo stage Ⅳ breast cancer treated in Tianjin Medical University Cancer Hospital and Cangzhou Hospital of Integrated TCM-WM from February 2010 to December 2017 were retrospectively analyzed. Related factors such as age, time of chief complaint, the clinical T/N stage, site of metastasis, expressions of molecular markers and treatment mode were included. Univariate log-rank test and multivariate Cox regression model were used to analyze the effects of prognostic factors on patients' overall survival (OS).Results:Univariate analysis showed that there were statistically significant differences in the OS of patients stratified by clinical N stage at first diagnosis, metastasis sites at first diagnosis, ER expression, progesterone receptor (PR) expression, Ki-67 positive index and p53 expression, endocrine therapy, chemotherapy at first diagnosis, surgery and radiotherapy of the primary lesions (all P < 0.01). Multivariate Cox regression analysis results showed that metastasis sites at first diagnosis, Ki-67 positive index, surgery and radiotherapy of the primary lesions were all independent influencing factors of OS for breast cancer patients (all P < 0.01). Conclusions:Patients with ER-positive de novo stage Ⅳ breast cancer have a good prognosis when they have oligometastasis, Ki-67 positive index ≤ 20%, and they receive surgery and radiotherapy of the primary lesions.

Application and thinking of working-process based CBL teaching method for undergraduate practice teaching in clinical laboratory of hematology / 中华医学教育探索杂志

Clinical laboratory of hematology is highly professionalized, with abstract concepts, and a variety of laboratory techniques. The working-process based CBL teaching method may help clinical laboratory interns better understand the clinical significance and the mutual relationship of laboratory tests, improve the comprehensive ability and cultivate the general clinical thinking ability.

Research on the scientific research performance evaluation of young scientists in hospitals based on Analytic Hierarchy Process and Data Envelopment Analysis / 中华医学科研管理杂志

Objective:To establish a performance evaluation index system and evaluation method for young scientists in hospitals, and to provide reliable evidence for management departments in evaluating, selecting outstanding young scientists and formulating tailored training programs.Methods:Literature analysis, expert consultation, analytic hierarchy process and data envelopment analysis were adopted in this article to evaluate the scientific research performance of young scientists in hospitals.Results:The scientific research data of young scientists under 45 years of age in Peking University Cancer Hospital from 2015 to 2019 were collected, and their scale efficiency and technical efficiency were analyzed by using index system and its weight and propose the improvement measures to achieve the purpose of evaluation and reform.Conclusions:This method is useful for the effective evaluation of the young scientists in the hospital, and is helpful to improve the level of scientific research management, as well as realize the virtuous circle of matching the input and output of scientific and technological innovation.

Exploratory study on the evaluation dimension and index of research hospital / 中华医学科研管理杂志

Objective:To explore the evaluation dimensions and indicators of research hospital, provide empirical evidence for the construction of research hospital.Methods:Non-probability sampling was adopted, physicians from 9 hospitals in Shanghai that have a National Clinical Medical Research Center or Shanghai Clinical Medical Research Center were invited as survey participants. The physician participants were divided into senior and junior groups. For senior group, a questionnaire including 5 dimensions and 21 indicators was provided. The survey participants were consulted to determine whether the indicators can be used to measure this dimension, and also invited to propose additional dimensions and indicators for improvement.For junior group, two open questions were proposed to explore their needs of support in both clinical service and research.Results:Based on suggestions from the research participants, the research team, and other expert consultant, this article tried to propose 5 dimensions and 21 indicators for evaluating research hospitals. Among them, 11 were indicators compiled by the research team based on the literature review and agreed by more than 2/3 of senior physicians surveyed, while others were proposed based on the survey results. The research team planned to use the entropy method to determine the weights of different indicators, thus, the participants were not required to make judgments on the weights of dimensions and indicators.Conclusions:The evaluation of research hospitals has not yet formed a systematic consensus. Through the exploration and establishment of evaluation dimensions and indicators, identify hospitals that are closer to the development goals of research hospitals can provide a basis for future next-step practices.

Identification of a super-spreading chain of transmission associated with COVID-19

BackgroundSuper-spreading events were associated with the outbreaks of SARS and MERS, but their association with the outbreak of COVID-19 remains unknown. Here, we report a super-spreading transmission chain of SARS-CoV-2 involving an index patient, seven cancer patients, 40 health care workers and four family members. MethodsWe conducted a retrospective study to identify the index patient and the exposed individuals linked to a chain of transmission associated with COVID-19. We collected and analyzed the data on demographic features, exposure history, clinical presentation, laboratory investigation, radiological examination, and disease outcome of these patients. ResultsWe identified the index patient and another presumptive "super-spreader", who initiated and amplified a super-spreading transmission chain associated with COVID-19, respectively. There were 31 female and 21 male patients in this cohort, and the median age was 37 years (range: 22-79 years). Each of them had an exposure history with the index patient or his close contacts. Approximately 87% (45/52) of the patients had fever or other symptoms, 96% (50/52) had abnormal chest CT-scan findings, 86% of the tested patients (39/45) were positive for SARS-CoV-2 in the nasopharyngeal or throat swab specimen, 85% of the tested patients (29/34) were positive for SARS-CoV-2-specific IgM and/or IgG, 15% of the RT-PCR positive patients were tested negative for the specific IgM and/or IgG at the convalescent phase, and 15% of the RT-PCR negative patients were tested positive for the specific IgM and/or IgG. The severe patients experienced a significant decrease in oximetry saturation, lymphocyte, and platelet counts, along with a significant increase in C-reactive protein, D-dimer, and lactate dehydrogenase. All six fatal cases had comorbidities and five of the seven cancer patients (71%) died within 2-20 days of the disease onset. ConclusionsThe super-spreading events were associated with the outbreak of COVID-19 in Wuhan and its impact on disease transmission warrants further investigation. Cancer patients appeared highly vulnerable to COVID-19. The finding that a significant portion of SARS-CoV-2 infected patients were tested negative for the serum specific IgM and IgG at the convalescent phase should be addressed by additional studies.

Development and Evaluation of A CRISPR-based Diagnostic For 2019-novel Coronavirus

BackgroundThe recent outbreak of infections by the 2019 novel coronavirus (2019-nCoV), the third zoonotic CoV has raised great public health concern. The demand for rapid and accurate diagnosis of this novel pathogen brought significant clinical and technological challenges. Currently, metagenomic next-generation sequencing (mNGS) and reverse-transcription PCR (RT-PCR) are the most widely used molecular diagnostics for 2019-nCoV. Methods2019-nCoV infections were confirmed in 52 specimens by mNGS. Genomic information was analyzed and used for the design and development of an isothermal, CRISPR-based diagnostic for the novel virus. The diagnostic performance of CRISPR-nCoV was assessed and also compared across three technology platforms (mNGS, RT-PCR and CRISPR) Results2019-nCoVs sequenced in our study were conserved with the Wuhan strain, and shared certain genetic similarity with SARS-CoV. A high degree of variation in the level of viral RNA was observed in clinical specimens. CRISPR-nCoV demonstrated a near single-copy sensitivity and great clinical sensitivity with a shorter turn-around time than RT-PCR. ConclusionCRISPR-nCoV presents as a promising diagnostic option for the emerging pathogen.

Advances in gene editing of Corynebacterium glutamate / 生物工程学报

Corynebacterium glutamicum, an important microorganism to produce amino acids and organic acids, has been widely applied in food and medicine fields. Therefore, using editing tools to study the function of unknown genes in C. glutamicum has great significance for systematic development of industrial strain with efficient and novel production capability. Recently, gene editing has been greatly developed. Traditional gene editing based on homologous recombination and gene editing mediated by nuclease are successfully applied in C. glutamicum. Among these, the CRISPR system has been developed to be a main tool used for gene knockout of C. glutamicum due to its advantages of efficiency, simplicity and good target specificity. However, more efficient and reliable knockout system is still urgently demanded, to help develop high-performing strains in industrial application.

Survival time of HIV/AIDS cases and influencing factors in Gansu province, 1997-2018 / 中华流行病学杂志

Objective@#To understand the survival time and influencing factors of HIV/AIDS cases in Gansu province from 1997 to 2018.@*Methods@#A retrospective cohort study was conducted to analyze the AIDS epidemic data of Gansu from 1997 to 2018 collected from the National HIV/AIDS information system. Life-span table were used to calculate survival rate, Kaplan-Meier method was used to draw the survival curves and calculate the average survival time, the Cox proportional hazard regression model were used to analyze the risk factors for death for HIV/AIDS cases.@*Results@#Among 6 813 HIV/AIDS cases, 715 (10.5%) died, and the average survival time was 195.9 months (95%CI: 189.7-202.2). The survival rates of 12 months, 60 months, 120 months and 180 months were 91.5%, 86.1%, 79.9% and 73.8%, respectively. Cox proportional hazard regression model showed that the risk factors for death in the HIV/AIDS cases were age (≥51 years old vs. ≤25 years old, HR=1.906, 95%CI: 1.353-2.685), transmission route (blood borne and others transmission vs. heterosexual transmission, HR=1.593, 95%CI: 1.226-2.069), detection way (hospital admission detection, blood transfusion and preoperative examination vs. entry-exit health examination, pre-marital examination and physical examination of recruits, HR=5.113, 95%CI: 2.083-12.547), disease phase (AIDS phase vs. HIV infection phase: HR=4.012, 95%CI: 3.401-4.732), baseline CD4 count (no CD4 detected vs. CD4 count ≥350/μl, HR=5.446, 95%CI: 3.835-7.732), antiretroviral therapy (receiving no antiretroviral therapy vs. receiving antiretroviral therapy, HR=12.019, 95%CI: 9.861-14.648).@*Conclusions@#The average survival time of HIV/AIDS cases was above 16 years in Gansu during 1997 to 2018. Death risk of HIV/AIDS cases might be increased by age ≥51 years, hospital admission detection, blood transfusion and preoperative examination, AIDS phase of disease phase, no baseline CD4 detected and no receiving antiretroviral therapy. It is necessary to conduct early HIV test, diagnosis and antiretroviral treatment and increase antiretroviral treatment rates and CD4 testing rate to improve the survival of HIV/AIDS cases.

Expression of SHIP 1 in the Patients with Acute Myeloid Leukemia and Its Influence on the Apoptosis of Human Leukemia Cells / 现代生物医学进展

Objective:To investigate the expression of SHIP1 in the patients with acute myeloid leukemia and its effect on the apoptosis of human leukemia cells.Methods:The expression of SHIP1 in the bone marrow of patients with acute myeloid leukemia was detected by Westem blot.U937 cells was transfected with SHIP1 expression vector (pEGFP-SHIP1 group) and empty vector control (pEGFP group) respectively,U937 cells without transfection were used as the control group.Flow cytometry was used to detect the apoptosis of the cells,the expression of SHIP1,Bcl-2,Bax,Akt,p-Akt were detected by western blot.Results:The expression of SHIP1 in the bone marrow of patients with acute myeloid leukemia was significantly lower than that of the normal human bone marrow SHIP 1 (P＜0.01).The SHIP1 and Bax expressions as well as the apoptotic rate ofpEGFP-SHIP1 group were significantly higher than those of the control group(P＜0.01),while the Bcl-2 and p-Akt expressions were significantly lower than those in the control group(P＜0.01).Conclusions:SH-P1 expression was down regulated in the bone marrow of patients with acute myeloid leukemia.SHIP1 could promote the apoptosis of human leukemia cells via Akt signaling pathway.

Clinical efficacy of apatinib in treating refractory triple-negative advanced breast cancer / 中国肿瘤临床

Objective:To evaluate the effectiveness and safety of using apatinib in the treatment of refractory triple-negative advanced breast cancer. Methods:Eight cases of advanced triple-negative breast cancer patients confirmed via histopathology, who were previously treated with anthracycline, taxane, gemcitabine, capecitabine, and 500 mg/d apatinib in our hospital from July 2015 to November 2016, were retrospectively analyzed. The time of disease progress, effective rate, clinical benefits, and side effects were observed. Results:Eight patients were administrated with an average of 4 treatment cycles, and the effects were evaluated after 2 weeks. Four patients exhibited partial remission, 3 had a stable disease, and 1 had a progressive disease. The disease control rate was 87.5%, and the median progression free survival was 4.2 months. The main side effects were hand-foot syndrome (3/8), bone marrow arrest (4/8), hypertension (2/8), proteinuria (3/8), hemoptysis (1/8), nausea (2/8), and fatigue (2/8). Most of these side effects were tolerable. Conclusion:Apatinib can effectively and tolerably prolong survival time and improve the quality of life of patients with advanced triple-negative breast cancer.

Tyrosine kinase inhibitors for the intervention of major morbid events in myeloproliferative neoplasms：reports from the 57th American Society of Hematology annual meeting / 白血病·淋巴瘤

With the research progress of pathogenesis of JAK gene in myeloproliferative neoplasms (MPN), more tyrosine kinase inhibitors were developed. MPN quantify scoring system is used to determine the efficacy of tyrosine kinase inhibitors for MPN. The choice of tyrosine kinase inhibitors, tyrosine kinase for the relief of MPN symptom burden, etc, become the topics of the 57th American Society of Hematology (ASH) annual meeting.

Sterile repositionable hemostasis clipping device in combination with high frequency electric snare in resection of colorectal polyps with wide and long peduncle / 中国内镜杂志

Objective To discuss the efficacy and safety of sterile repositionable hemostasis clipping device in combination with high frequency electric snare in polypectomy of colorectal polyps with wide and long peduncle. Methods From January 2014 to December 2015, 21 cases of colorectal polyps with wide and long peduncle (diameter greater than 2 cm) in endoscopic treatment under electronic colonoscopy. We used 1 ~ 2 hemostatic clips to clip colorectal polyps roots, then used electric resection with high frequency electric snare electric coagulation. Postoperative bleeding, perforation were observed follow-up. Results 24 polyps in 21 cases were removed one-time successfully. Stump errhysis in 1 case, hot biopsy forceps is given to deal with local wound followed by Olympus Clip HX-610-135L EZ titanium clip. There was no complication such as bleeding and perforation in 3 to 6 months after the operation. In the colonoscopy examination, recurrence of polyps were not found in the original polyp resection site. Conclusion Sterile repositionable hemostasis clipping device in combination with high frequency electric snare in polypectomy of colorectal polyps with wide and long peduncle is safe and effective, without bleeding or perforation.

A study of immunocyte subsets and serum cytokine profiles before and after immunal suppression treatment in patients with immune thrombocytopenia / 中华内科杂志

Objective To explore the clinical significance of a series of cytokines and peripheral blood immunocyte subsets before and after immunosuppressive therapy in patients with immune thrombocytopenia (ITP).Methods The percentages of immunocyte subsets in the peripheral blood of 20 patients with ITP and 20 healthy controls were detected by flow cytometry,including CD3+,CD4+,CD8+,CD4+/CD8+,CD1~.ELISA was applied to detect the level of serum TNFα,IL-2,IL-6,IL-4,IL-10,IL-11,IL-17,IL-27,transforming growth factor β (TGFβ),thrombopoietin (TPO) of 20 patients with ITP and 20 healthy controls.Results The percentage of CD3+ T lymphocyte,CD4+ T lymphocyte and the ratio of CD4+ / CD8+ T lymphocyte in patients with ITP were lower than those in healthy controls [(62.66 ± 6.58) ％ vs (69.93 ± 4.81) ％,(29.46 ± 5.02) ％ vs (39.08 ± 3.50) ％,0.97 ± 0.35 vs 1.56 ± 0.26,all P ＜ 0.05].After immunosuppressive therapy,the percentage of CD3+ T lymphocyte,CD4+ T lymphocyte and the ratio of CD4+/CD8+ T lymphocyte [(71.49 ±5.16)％,(39.25 ±3.21)％ and 1.56 ±0.28] recovered to the same levels in healthy controls.The percentage of CD8+ T lymphocyte and CD19+ B lymphocyte in patients with ITP were higher than those in the healthy controls [(30.28 ±4.63)％ vs (25.90±3.06)％,(18.92 ± 4.27)％ vs (13.17 ± 3.64)％,all P ＜ 0.05].After treatment of immunosuppressive therapy,the percentage of CD8+ T lymphocyte and CD19+ B lymphocyte [(25.16 ± 3.45) ％ and (11.98 ± 3.68) ％] recovered to the similar levels in healthy controls.The serum levels of IL-4,IL-6,IL-11,IL-17 and TPO in patients with ITP were significantly higher than those in healthy controls.While TGFβ level was significantly decreased.There was no significant difference of IL-27 between ITP patients and healthy controls.After the treatment of immunosuppressive therapy,IL-4,IL-6,IL-11,IL-17,TPO and TGFβ were down-regulated while IL-27 was up-regulated.There was no significant difference of IFNγ,TNFα,IL-2 and IL-10 among ITP patients before or after immunosuppressive therapy and healthy controls.Conclusions The present study suggests that the aberrant immunocyte subsets and cytokines are involved in the pathogenesis of ITP.Hyper-function of Th2 and Th17,dysfunction of Treg cells,up-regulation of IL-27,IL-11,TPO and other factors may contribute to the pathogenesis of ITP.

Diagnostic value of serum AFP-L3, GP73 and GGT combined detection in hepatocellular carcinoma / 肿瘤研究与临床

Objective To explore the clinical value of AFP-L3,GP73 and GGT as biomarkers in diagnosis of hepatocellular carcinoma (HCC).Methods According to the pathological diagnosis,141 patients were divided into two groups,HCC group were 74 cases,benign liver disease group were 67 cases.Use ELISA method tested the serum AFP-L3 and GP73 levels.The GGT level was detected by the automatic biochemical instrument of all the 141 patients.AFP-L3,GP73 and GGT concentration difference was compared between the two groups.ROC curve was used to determine the cut-off level to diagnose HCC.The value of single use AFP-L3,GP73,GGT and joint the three indexes to diagnose HCC were analyzed.Results The average level of AFP-L3 in the patients with HCC was (113.58±63.62) μg/L,it was significantly higher than that in the patients with benign liver diseases [(23.19±34.54) μg/L] (P ＜ 0.001).The area under the ROC curve of AFP-L3 level was 0.802.Taking AFP-L3 level ≥ 38.47 μg/L as diagnostic criteria,the sensitivity of AFP-L3 level in HCC diagnosis was 81.08 ％ and the specificity was 88.06 ％.The average level of GP73 in the patients with HCC was (126.55±49.56) μg/L,it was significantly higher than that in the patients with benign liver diseases [(56.97±26.48) μg/L] (P ＜ 0.001).The area under the ROC curve of GP73 level was 0.811.Taking GP73 level≥69.44 μg/L as diagnostic criteria,the sensitivity of GP73 level in HCC diagnosis was 75.68 ％ and the specificity was 91.04 ％.The average level of GGT in the patients with HCC was (173.20±179.18) U/L,it was significantly higher than that in the patients with benign liver diseases [(90.77±81.53) U/L] (P ＜ 0.001).The area under the ROC curve of GGT level was 0.713.Taking GGT level ≥ 110.77 U/L as diagnostic criteria,the sensitivity of GGT level in HCC diagnosis was 74.32 ％ and the specificity was 77.61％.Joint use AFP-L3,GP73 and GGT to diagnose HCC,the sensitivity was 83.78 ％,specificity was 92.53 ％.Conclusion Combined detection of tumor markers AFP-L3,GP73 and GGT can improve the positive rate of HCC,which has good clinical application value.

The relationship of IL-11 and CTGF expression with the bone metastasis of breast cancer / 中华检验医学杂志

Objective To investigate the role of IL-11 and CTGF in bone metastasis of breast cancer.Methods A total of 180 pathologically confirmed breast cancer patients in Tianjin Medical University Cancer Institute and Hospital were enrolled,90 of which had bone metastasis.Twenty healthy people who took physical examination at the same period were adopted as controls,excluding those with endocrine or metabolic disease or other chronic diseases.Peripheral blood samples were collected and ELISA was employed to detect IL-11 and CTGF expression.Forty paraffin-embedded breast cancer tissue sections from those with bone metastasis and forty tissue sectioned from those without bone metastasis were detected for IL-11 and CTGF expression with immunohistochemisty.Results Serum IL-11 level was (242.9 ±56.3) μg/L in the group with bone metastasis and (85.9 ± 35.7) μg/L in the group without bone metastasis,with a significant difference (F =43.532,P ＜0.01).Serum level of CTGF was (15.6 ±7.4) μg/L in the group with bone metastasis and (15.0 ± 7.0) μg/L in the group without bone metastasis,with no significant difference (F =3.007,P ＞ 0.05).The rate of positive immunohistochemical staining for IL-11 in the group with bone metastasis (57.5％) was significantly higher than that in the group without bone metastasis (14.3％) (x2 ＝ 36.626,P ＜ 0.01).There was no significant difference in the positive rate of CTGF expression between the group with bone metastasis (17.5％) and the group without metastasis (14.3％) (x2 =0.370,P ＞ 0.05).Conclusions IL-11 expression is correlated with bone metastasis of breast cancer.Breast cancer patients with high IL-11 expression are more prone to develop bone metastasis.

Pharmacoeconomic analysis of paclitaxel plus epirubicin or carboplatin for metastatic breast cancer / 中国肿瘤临床

Objective:This study evaluated the economical effects of two different regimens for metastatic breast cancer, namely, paclitaxel plus either carboplatin (TP) or epirubicin (TE). Methods:The cost-effectiveness method in pharmacoeconomics was adopted to analyze retrospectively the two different regimens. Results:The median follow up was 23.5 (range:9 to 42) months. The overall response rate for TP and TE were 78.33%and 80.00%, respectively. The 1-and 2-year progression-free survival rates of TP and TE were 43.6%and 38.9%and 10.8%and 17.4%, respectively. The 1-and 2-year overall survival rates were 80.3%and 78.3%, respectively for TP, whereas the corresponding values for TE were 53.2%and 47.9%. No statistically significant difference was found between the two groups (P>0.05). Cost-effectiveness analysis showed that the average costs of the TP and TE regimens were 10 303.8 and 13 853.3 yuan, respectively, with corresponding cost-effectiveness ratios of 131.54 and 173.17 (P<0.01). For the chemotherapy toxicity, the alopecia reactions of the TP group were significantly lower than those of the TE group (P<0.01). Conclusion:The short-and long-term efficacies of the two regimens were similar. TP regimen was the optimal scheme for advanced metastatic breast cancer.

Intelligent optimization of the structure of the large section highway tunnel based on improved immune genetic algorithm / 药物分析学报

As in the building of deep buried long tunnels, there are complicated conditions such as great deformation, high stress, multi-variables, high non-linearity and so on, the algorithm for structure optimization and its application in tunnel engineering are still in the starting stage. Along with the rapid development of highways across the country, It has become a very urgent task to be tackled to carry out the optimization design of the structure of the section of the tunnel to lessen excavation workload and to reinforce the support. Artificial intelligence demonstrates an extremely strong capability of identifying, expressing and disposing such kind of multiple variables and complicated non- linear relations. In this paper, a comprehensive consideration of the strategy of the selection and updating of the concentration and adaptability of the immune algorithm is made to replace the selection mode in the original genetic algorithm which depends simply on the adaptability value. Such an algorithm has the advantages of both the immune algorithm and the genetic algorithm, thus serving the purpose of not only enhancing the individual adaptability but maintaining the individual diversity as well. By use of the identifying function of the antigen memory, the global search capability of the immune genetic algorithm is raised, thereby avoiding the occurrence of the premature phenomenon. By optimizing the structure of the section of the Huayuan tunnel, the current excavation area and support design are adjusted. A conclusion with applicable value is arrived at. At a higher computational speed and a higher efficiency, the current method is verified to have advantages in the optimization computation of the tunnel project. This also suggests that the application of the immune genetic algorithm has a practical significance to the stability assessment and informationlzation design of the wall rock of the tunnel.

Effects of erythropoietin on proliferation and cell cycle of human bone marrow-derived mesenchymal stem cells / 中国组织工程研究

BACKGROUND: Researches show that erythropoietin (EPO) can stimulate the proliferation and protraction of endothelial progenitor cells to form new vessels, so EPO may play an important role in proliferation and differentiation of bone marrow-derived mesenchymal stem cells (MSCs). OBJECTIVE: To explore the effects of EPO on the proliferation and cell cycle of MSCs. DESIGN, TIME AND SETTING: From July to November 2007, the observation of comparative cell trial was performed at the Hematologic Diseases Institute of General Hospital Lanzhou Military Area Command of Chinese PLA. MATERIALS: Bone marrow liquid specimens were provided by voluntary donors. The informed consents were obtained from all patients, and the experiment was approved by Hospital Ethics Committee. METHODS: Using Percoll solution, MSCs were isolated from bone marrow by the method of density centrifugation. Flow cytometry was applied to detect the cell cycles of the second and third passages. MSCs were incubated with different doses of EPO (0.25, 0.50, 1.00, 2.00, 5.00 U/mL) in serum free culture media, and cells cultured with no EPO were regarded as control. All cells were cultured for 24, 48, and 72 hours. MAIN OUTCOME MEASURES: ①Proliferation of MSCs was measured by MTT assays after 24, 48, and 72 hours; ②Cell cycle was measured by flow cytometry assays after incubated with 10 U/mL EPO for 72 hours. RESULTS: After MSCs was incubated with EPO, the cell proliferation index was significantly increased in a dose and time dependent manner. The effects on the proliferation of MSCs were highest in 5 U/mL group. Compared with the control group, EPO could significantly decrease G0 /G1 ratio, and increase S and G2/M stage ratio (P

Identification and in vitro BrdU labeling of adult bone marrow mesenchymal stem cells / 中国组织工程研究

BACKGROUND: No particular marker molecule of bone marrow mesenchymal stem cells (BMSCs) is presently found, so its determination is difficult. BrdU marker has no radioactive pollution. Some studies have confirmed that BrdU marker has no damage to cell function without ultraviolet radiation. OBJECTIVE: To investigate in vitro identification and labeling methods and biological characteristics of adult BMSCs. DESIGN, TIME AND SETTING: The cell experiment was conducted at the General Hospital of Lanzhou Military Area Command of Chinese PLA between June and December 2007. MATERIALS: Bone marrow was collected from 5 healthy adult volunteers. BrdU was purchased from Sigma, USA. METHODS: 10 mL adult bone marrow was harvested to isolate mononuclear cells by density gradient. Cells were cultured in DMEM containing 10% fetal bovine serum and proliferated at a density of 2?108 L-1. At the third passage, BMSCs was inoculated in medium supplemented with osteoblast inductor and stained with alkaline phosphatase. Subsequently, BMSCs were inoculated in medium supplemented with lipoblast inductor and stained with oil red O. Cells were incubated with BrdU at different concentrations of 5, 10 and 15 ?mol/L for 12, 24, 48, 72 and 96 hours, and then detected by immunocytochemistry. MAIN OUTCOME MEASURES: Cell growth and proliferation were observed under an inverted light microscope. Cell phenotype, osteoblast and lipoblast differentiation were identified by flow cytometry. BrdU-positive labeling rate and cell growth after labeling were investigated. RESULTS: In vitro cultured BMSCs were homogenous population and exhibited a spindle-shaped fibroblastic morphology. BMSCs expressed CD44 and CD71, but did not express CD34 and HLA-DR. BMSCs can differentiate into osteoblasts and lipoblasts. Most BMSCs were labeled by BrdU. With the increase in labeling concentration and over time, BrdU positive rate gradually increased and exceeded 90% after labeling with BrdU (10 ?mol/L) for 72 hours, with the labeling identifiable in nine consecutive passages. At the third passage, 90% BMSCs were in G0/G1 phase, and 88.62% in G0/G1 phase after labeling. BrdU has little effect on morphous and proliferation of labeled cells. CONCLUSION: Adult BMSCs can be identified through morphological character, specific surface antigens and multipotential differentiation in vitro. BrdU labeling provides a feasible means for a dynamic observation of the survival, growth and differentiation of the implanted BMSCs. The optimal dosage and timing of BrdU labeling are respectively 10 ?mol/L and 72 hours.