Comparison of mouse models of depression induced by different modeling methods / 生理学报

The present article was aimed to compare the effectiveness of different induction methods for depression models. Kunming mice were randomly divided into chronic unpredictable mild stress (CUMS) group, corticosterone (CORT) group, and CUMS+CORT (CC) group. The CUMS group received CUMS stimulation for 4 weeks, and the CORT group received subcutaneous injection of 20 mg/kg CORT into the groin every day for 3 weeks. The CC group received both CUMS stimulation and CORT administration. Each group was assigned a control group. After modeling, forced swimming test (FST), tail suspension test (TST) and sucrose preference test (SPT) were used to detect the behavioral changes of mice, and the serum levels of brain-derived neurotrophic factor (BDNF), 5-hydroxytryptamine (5-HT) and CORT were detected with ELISA kits. Attenuated total refraction (ATR) spectra of mouse serum were collected and analyzed. HE staining was used to detect morphological changes in mouse brain tissue. The results showed that the weight of model mice from the CUMS and CC groups decreased significantly. There was no significant change in immobility time of model mice from the three groups in FST and TST, while the glucose preference of model mice from the CUMS and CC groups was significantly reduced (P < 0.05). The serum 5-HT levels of model mice from the CORT and CC groups were significantly reduced, while the serum BDNF and CORT levels of model mice from the CUMS, CORT, and CC groups showed no significant changes. Compared with their respective control groups, the three groups showed no significant difference in the one-dimensional spectrum of serum ATR. The difference spectrum analysis results of the first derivative of the spectrogram showed that the CORT group had the greatest difference from its respective control group, followed by the CUMS group. The structures of hippocampus in the model mice from the three groups were all destroyed. These results suggest that both CORT and CC treatments can successfully construct a depression model, and the CORT model is more effective than the CC model. Therefore, CORT induction can be used to establish a depression model in Kunming mice.

Resuscitative endovascular balloon occlusion of the aorta in the treatment of severe hemorrhagic shock caused by upper gastrointestinal bleeding / 世界急诊医学杂志（英文）

@#Hemorrhagic shock is a life-threatening disease often encountered in emergency departments (EDs). Hemorrhagic shock caused by extensive bleeding from multiple sites is often associated with high mortality and morbidity. In recent years, resuscitative endovascular balloon occlusion of the aorta (REBOA) has been widely used in traumatic hemorrhagic shock and is considered to be an effective resuscitation measure.[1] Some studies reported that REBOA was also effective for non-traumatic hemorrhage.[2,3] In this study, we report a case of hemorrhagic shock caused by acute upper gastrointestinal bleeding that was successfully treated and received REBOA to obtain a transition time. This report may provide feasible options for emergency physicians, gastroenterologists, or surgeons to more actively treat refractory gastrointestinal bleeding.

Clinicopathlogic features and prognosis of the gastric signet ring cell carcinoma: a SEER database analysis / 医学研究生学报

Objective　Prognoses of late stage signet-ring cell carcinoma (SRCC) is usually worse than that in other gastric carcinoma. In the current study, SEER database were adopted to analyze the clinicopathologic features and prognoses of SRCC and non-signet-ring cell carcinoma (non SRCC), and to compare the differences in survival rate under different early treatments.　Methods　Clinical data of 5193 patients who were diagnosed with gastric carcinoma from 2010 to 2015 are collected from SEER. Patients are divided into two groups SRCC (n=2439) and non SRCC (n=2754) based on their histologic type. Differences in Gender, age, race, primary site, degree of differentiation, tumor size, depth of invasion, local lymph node metastasis, distant metastasis, tumor, node, metastasis (TNM) staging, operative treatment and chemotherapy were compared. Cox regression model was used to analyze prognostic factors. According to different operative treatment, patients in SRCC and non SRCC groups were divided into surgery group and surgery with adjuvant chemotherapy group, respectively. Kaplan-meier method was used to draw the survival curve, and Log-Rank test was adopted for survival analysis.　Results　Significant statistical difference (P<0.05) were found in the two gastric carcinoma groups regarding gender, age, race, primary site, degree of differentiation, tumor size, depth of invasion, local lymph node metastasis, distant metastasis, TNM staging, operative treatment and chemotherapy. The multivariate Cox regression analysis indicated that age (HR:1.417; 95%CI:1.273-1.578; P<0.05), race (HR:0.91; 95%CI:0.825-0.998; P<0.05), tumor size (HR:1.28; 95%CI: 1.199-1.365; P<0.05), depth of invasion (HR: 1.252: 95%CI: 1.159-1.352; P<0.05), local lymph node metastasis (HR:0.862; 95%CI: 0.81-0.918; P<0.05), distant metastasis (HR: 1.369: 95%CI: 1.069-1.753; P<0.05), TNM stage (HR:1.342; 95%CI:1.155-1.559; P<0.05), and surgical treatment (HR:0.245; 95%CI: 0.228-0.284; P<0.05) are independent risk factors affecting the prognosis of SRCC patients. The overall five-year survival rate of SRCC patients is 27.6% , which is lower than that of non SRCC patients (43.0%). Therefore, there is significant difference in statistics (P<0.05). Significant statistical difference was also found in stratification analysis of the five-year survival rates among SRCC surgery group, SRCC surgery with adjuvant chemotherapy group, non SRCC surgery group and non SRCC surgery with adjuvant chemotherapy group. The results indicated that the five-year survival rates of SRCC surgery with adjuvant chemotherapy group at stage TIN1M0 and stage T2N0M0 are both superior to that in the surgery group with statistical difference (P<0.05). In addition, the five-year survival rate of SRCC surgery with adjuvant chemotherapy group at T2N0M0 is superior to that in the non SRCC patients, with statistical difference (P<0.05).　Conclusion　SRCC patients present with unique clinicopathologic features. Early detection and treatment could improve the prognosis of SRCC patients.

Progress in diagnosis and treatment of medium chain acyl coenzyme A dehydrogenase deficiency / 中国实用儿科杂志

Medium chain acyl CoA dehydrogenase deficiency is a mitochondrial fatty acid oxidative deficiency disease. It has various clinical manifestations,such as hypoglycemia,lethargy,myasthenia,etc. Different clinical manifestations and atypical biochemical examination can increase the difficulty of diagnosis,which is more likely to result in misdiagnosis. If it is not treated in time,mortality and the rate of sequelae are high,but if confirmed by neonatal screening and treated in time,satisfactory results can be obtained.

Cardiac Fibroblast-Specific Activating Transcription Factor 3 Promotes Myocardial Repair after Myocardial Infarction / 中华医学杂志(英文版)

<p><b>Background</b>Myocardial ischemia injury is one of the leading causes of death and disability worldwide. Cardiac fibroblasts (CFs) have central roles in modulating cardiac function under pathophysiological conditions. Activating transcription factor 3 (ATF3) plays a self-protective role in counteracting CF dysfunction. However, the precise function of CF-specific ATF3 during myocardial infarction (MI) injury/repair remains incompletely understood. The aim of this study was to determine whether CF-specific ATF3 affected cardiac repair after MI.</p><p><b>Methods</b>Fifteen male C57BL/6 wild-type mice were performed with MI operation to observe the expression of ATF3 at 0, 0.5, 1.0, 3.0, and 7.0 days postoperation. Model for MI was constructed in ATF3TGfl/flCol1a2-Cre+ (CF-specific ATF3 overexpression group, n = 5) and ATF3TGfl/flCol1a2-Cre- male mice (without CF-specific ATF3 overexpression group, n = 5). In addition, five mice of ATF3TGfl/flCol1a2-Cre+ and ATF3TGfl/flCol1a2-Cre- were subjected to sham MI operation. Heart function was detected by ultrasound and left ventricular remodeling was observed by Masson staining (myocardial fibrosis area was detected by blue collagen deposition area) at the 28 day after MI surgery in ATF3TGfl/flCol1a2-Cre+ and ATF3TGfl/flCol1a2-Cre- mice received sham or MI operation. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect cell proliferation/cell cycle-related gene expression in cardiac tissue. BrdU staining was used to detect fibroblast proliferation.</p><p><b>Results</b>After establishment of an MI model, we found that ATF3 proteins were increased in the heart of mice after MI surgery and dominantly expressed in CFs. Genetic overexpression of ATF3 in CFs (ATF3TGfl/flCol1a2-Cre+ group) resulted in an improvement in the heart function as indicated by increased cardiac ejection fraction (41.0% vs. 30.5%, t = 8.610, P = 0.001) and increased fractional shortening (26.8% vs. 18.1%, t = 7.173, P = 0.002), which was accompanied by a decrease in cardiac scar area (23.1% vs. 11.0%, t = 8.610, P = 0.001). qRT-PCR analysis of CFs isolated from ATF3TGfl/flCol1a2-Cre+ and ATF3TGfl/flCol1a2-Cre- ischemic hearts revealed a distinct transcriptional profile in ATF3-overexpressing CFs, displaying pro-proliferation properties. BrdU-positive cells significantly increased in ATF3-overexpressing CFs than control CFs under angiotensin II stimuli (11.5% vs. 6.8%, t = 31.599, P = 0.001) or serum stimuli (31.6% vs. 20.1%, t = 31.599, P = 0.001). The 5(6)-carboxyfluorescein N-hydroxysuccinimidyl ester assay showed that the cell numbers of the P2 and P3 generations were higher in the ATF3-overexpressing CFs at 24 h (P2: 91.6% vs. 71.8%, t = 8.465, P = 0.015) and 48 h (P3: 81.6% vs. 51.1%, t = 9.029, P = 0.012) after serum stimulation. Notably, ATF3 overexpression-induced CF proliferation was clearly increased in the heart after MI injury.</p><p><b>Conclusions</b>We identify that CF-specific ATF3 might contribute to be MI repair through upregulating the expression of cell cycle/proliferation-related genes and enhancing cell proliferation.</p>

Analysis of related parameters of ocular biometric values in children and adolescents / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:To measure ocular biometric values with sexual and age and determine the relationship between the differences using the Lenstar 900.METHODS:Totally 413 myopes 826 eyes,200 males (400 eyes) and 213 females (426 eyes),were enrolled in this study and were divided into 3 groups:Group Ⅰ (＜ 5 years),Group Ⅱ (5-10 years),Group Ⅲ (＞10 years).Central corneal thickness (CCT),anterior chamber depth (ACD),lens thickness (LT),axial length (AL),white-to-white distance (WWD) and pupil diameter (PD) were measured by Lenstar 900.The differences between age groups and gender groups were compared using the LSD and SNKk methods in variance analysis.Pearson correlation coefficient to assess AL,CCT,ACD,LT,WWD,PD in children and adolescents.RESULTS:There were significant difference in CCT between ages groups (P＜0.05) which increased with the age.There were significant differences both in ACD and AL between sexual groups.With analysis of Person,CCT showed a significantly positive correlation with WWD and PD(r=0.208,0.167;P＜0.05) and ACD showed a significantly positive correlation with AL,WWD,PD(r=0.620,0.238,0.192;P＜0.05).LT showed a significantly negative correlation with ACD,AL and WWD (r=-0.271,-0.186,-0.227;P＜0.05).WWD showed a significantly positive correlation with PD (r=0.273,P＜0.05).CONCLUSION:CCT has gradually thickening trend with ages.Men are more than women in ACD and AL.CCT shows positive correlation with WWD and PD and ACD shows positive correlation with AL,WND,PD.LT shows negative correlation with ACD,AL and WWD.WWD showed positive correlation with PD.

Application of high-content screening and flow cytometry analysis techniques to evaluation of myocardial fibroblasts proliferation / 生理学报

The proliferation of cardiac fibroblasts (CFs) is a key pathological process in the cardiac remodeling. To establish an objective, quantitative method for the analysis of cell proliferation and cell cycle, we applied the high-content screening (HCS) and flow cytometry (FCM) techniques. CFs, isolated by enzyme digestion from newborn C57BL/6J mice, were serum starved for 12 h and then given 10% fetal bovine serum (FBS) for 24 h. Followed by BrdU and DAPI (or 7-AAD) staining, CFs proliferation and cell cycle were analyzed by HCS and FCM, respectively. Discoidin domain receptor 2 (DDR2) staining indicated that the purity of isolated CFs was over 95%. (1) HCS analysis showed that the ratio of BrdU-positive cells was significantly increased in 10% FBS treated group compared with that in serum-free control group [(12.96 ± 0.67)% vs (2.77 ± 0.33)%; P < 0.05]. Cell cycle analysis showed that CFs in G0/G1 phase were diploid, and CFs in S phase were companied with proliferation, DNA replication and enlarged nuclei; CFs in G2 phase were tetraploid, and CFs in M phase produced two identical cells (2N). (2) FCM analysis showed that the ratio of BrdU-positive cells was increased in 10% FBS treated group compared with that in the control group [(11.10 ± 0.42)% vs (2.22 ± 0.31)%; P < 0.05]; DNA content histogram of cell cycle analysis indicated that the platform of S phase elevated in 10% FBS group compared with control group. (3) There were no differences between the two methods in the results of proliferation and cell cycle analysis. In conclusion, HCS and FCM methods are reliable, stable and consistent in assessment of the proliferation and cell cycle in CFs.

Inhibition of p38 activity reverses claudin-6 induced cell apoptosis, invasion, and migration / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Claudin-6 is a protein component of tight junctions and its expression could downregulate the malignant phenotype of breast carcinoma. Here we investigated the mechanisms of claudin-6 induced human MCF-7 breast cancer cells apoptosis, invasion, and migration.</p><p><b>METHODS</b>Terminal deoxyribonucleotide transferase-mediated nick-end labeling assay and Annexin-V/PI double stain assay were carried out to evaluate apoptosis. Inhibitors of each pathway were used to inactivate the signaling pathways. The expression of claudin-6 and phosphate p38, Erk 1/2 and Akt protein levels was confirmed by Western blotting analysis. Invasive and migratory traits of claudin-6 expressing cells were determined by Boyden chamber invasion assay and monolayer wound-healing assay.</p><p><b>RESULTS</b>Cells with high-level expression of claudin-6 had a higher rate of apoptosis than control cells. Western blotting assay showed that by contrast to control groups, p38 pathways were more activated in claudin-6 expressing cells. However, after inhibitor SB203580 treatment, the activation status could be significantly counteracted. Furthermore, by applying inhibitors to the apoptotic rate, invasive and migratory traits were also recovered in cells with claudin-6 expression.</p><p><b>CONCLUSION</b>Claudin-6 may function through p38 mitogen-activated protein kinase pathway, of which inhibition may reverse claudin-6-induced cell apoptosis, invasion, and migration.</p>

Fluorouracil implants for colorectal cancer: a systematic review and meta-analysis / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To evaluate the benefit and safety of fluorouracil implants on colorectal cancer.</p><p><b>METHODS</b>Based on the methods of Cochrane systematic reviews, databases including CBM(1982 to March 2011), CNKI(1911 to March 2011), EMBASE(1966 to March 2011), and Medline(1950 to March 2011) were searched to identify randomized controlled trials assessing the benefit of fluorouracil implants on colorectal cancer. The quality of the included studies was assessed using the Cochrane's tool for assessing bias. RevMan5.0 was used for meta-analysis.</p><p><b>RESULTS</b>Sixteen studies were included(n=1223). The quality of included studies was moderate. Fluorouracil implants could reduce the 2-year mortality(RR=0.33. 95% CI:0.18-0.59), 2-year metastasis rate(RR=0.35, 95% CI: 0.19-0.66), and 2-year recurrence rate(RR=0.48, 95% CI:0.36-0.65). There were no significant differences in complications and adverse effects between fluorouracil implants and the control group.</p><p><b>CONCLUSIONS</b>Current evidence demonstrates that fluorouracil implants may modestly improve the outcome of colorectal cancer patients without increasing its adverse events. However, the results should be interpreted with caution due to the risk of bias of included studies.</p>

Differential expression of proteins in rat mesenchymal stem cells undergoing endothelial differentiation / 中华血液学杂志

<p><b>OBJECTIVE</b>To screen and identify differentially expressed proteins of mesenchymal stem cells (MSC) during endothelial differentiation.</p><p><b>METHODS</b>MSCs were induced to endothelial differentiation with vascular endothelial growth factor (VEGF) and epithelial growth factor (EGF) mixture. The whole cell proteins were extracted and isolated by two-dimensional gel electrophoresis. After gel was analyzed by Imagemaster 5.0 software, differentially expressed proteins were partially selected and identified by MALDI-TOF-MS.</p><p><b>RESULTS</b>The differentiated MSC highly expressed endothelial cells related markers, CD31, CD34 and FVIIIAg were 56.8%, 38.8% and 14.5% respectively by flow cytometer. Compared with the primary cultured MSC, the differentiated cells differentially expressed 91 proteins. Among the 19 identified proteins, 11 up-regulated and 8 down-regulated, which include cytoskeletal proteins, such as myosin, filamin, vimentin and vinculin; cell metabolism enzymes, such as ORP-150, ERO1-α, Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, protein disulfide-isomerase A3, FAS and enolase 3; nuclear factors, such as TAR DNA binding protein, guanine nucleotide binding protein and hypoxia up-regulated protein 1; VEGF receptors, such as KDR and so on.</p><p><b>CONCLUSIONS</b>Cytoskeletal proteins, metabolism enzymes and KDR were all involved in endothelial differentiation of MSC. These proteins may be the regulatory targets for endothelial differentiation of MSC.</p>

Directional differentiation of human adipose-derived stem cells into endothelial cells / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the possibility of directional differentiation of human adipose stem cells (hADSCs) into endothelial cells (EC), so as to provide seed cells for tissue engineered vessels.</p><p><b>METHODS</b>hADSCs were isolated from human adipose tissue by collagenase digestion, cultured and amplified by adherence to flasks. Then hADSCs were directionally induced to differentiate into EC by a combination of fibronectin (FN), endothelial cells support liquid (EGM2-MV) containing various growth factors and high concentration of VEGF165 (50 ng/ml). Then, the cells morphology, phenotype and function were identified.</p><p><b>RESULTS</b>Highly homologous hADSCs were obtained, and then hADSCs were directionally differentiated into EC. CD31 and CD34, the specific markers for EC, and vascular endothelial growth factor receptor (KDR) were positive by immunohistochemical staining and RT-PCR. In addition, unique Weibel-Palade bodies in EC were observed under transmission electron microscope. Functionally, hADSCs could swallow Dil-Ac-LDL and form tube-like structures in matrigel after endothelial differentiation.</p><p><b>CONCLUSIONS</b>hADSCs can be successfully induced to differentiate into endothelial cells in vitro.</p>

Comparative study of whole blood lysis reagents for analysis of immunocytes in peripheral blood of mice by flow cytometry / 中国实验血液学杂志

This study was purposed to investigate the efficacy of different whole flow lysis reagents for lysis of red blood cells in flow cytometric analysis. The expression of immunocytes was detected by flow cytometry after lysis of red blood cells using commercial reagents (Optilyse C, FACS Lysing Solution) and self-made red blood cell lysis reagents (RBC Lysis Buffer), the detection results were analyzed comparatively. The results showed that there was no significant difference in the percentage of CD3e(+), CD3e(+)CD4(+), CD3e(+)CD8a(+), CD3e(-)CD19(+), CD3e(-)NK1.1(+) and Gr-1(+) cells between 3 different lysis reagent groups. However OptiLyse C solution was suitable to Gr-1(+) cell detection, but did not suit to Foxp3(+) Treg detection. The self-made RBC Lysis Buffer and FACS Lysing Solution were suited to Foxp3(+) Treg detection. It is concluded that the use of self-made RBC Lysis Buffer for flow cytometry can get the lysis efficiency of commercially available lysis solutions when samples are prepared in accordance with standardized procedure. The self-made RBC Lysis Buffer not only can satisfy experimental requirements, but also can reduce the experimental costs.

Gene therapy based on inducible IL-12 gene in a mouse model of orthotopically transplanted H22 hepatoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To evaluate the antitumor efficiency of IL-12 gene induced by RU486 regulatory system in a mouse model of orthotopically transplanted hepatoma.</p><p><b>METHODS</b>The orthotopic hepatoma model was prepared by inoculation of H22 hepatoma cells into the mouse liver. Murine interleukin-12 (IL-12) expressing plasmid pRS22 containing RU486 regulatory system was injected into mice by a hydrodynamic injection 3 days after H22 cells inoculation. Three days after hydrodynamic injection, the mice were induced with RU486 (250 µg/kg) consecutive intraperitoneal administration for 6 days. Blood samples were taken at 10 h after the first and third induction for the determination of IL-12, IFN-γ and NO. Five mice were sacrificed at 2 days after the treatment with RU486. The tumor size was measured. HE and immunohistochemical stainings were applied to evaluate the proliferative activity and angiogenesis in the tumors. The other 7 mice were kept to monitor their survival.</p><p><b>RESULTS</b>In mice receiving saline plus RU486, pRS-LacZ plus RU486, or pRS22 plus sesame oil, the liver tumors were big in size: (409.90 ± 137.03) mm(3), (271.80 ± 182.63) mm(3) and (251.00 ± 76.55) mm(3), respectively. Strong PCNA positive expression [(82.10 ± 4.62)%, (83.45 ± 2.34)% and (77.46 ± 2.99)%] and extensive microvessel density (74.58 ± 18.47, 63.60 ± 13.36 and 53.52 ± 11.74 per 400 × field), respectively, in these tumor tissues were observed after immunohistochemical staining. The survival period was shorter in these mice. In contrast, in mice treated with pRS22 plus RU486, the tumor was smaller in size. Extensive necrosis, weak PCNA proliferative activity (50.67 ± 8.09)%, and a marked paucity of microvessel density (25.38 ± 10.87) were seen. The survival of mice was obviously prolonged. Compared with the 3 control groups, a significant elevation of serum IL-12, IFN-γ and NO levels were detected in the mice treated with pRS22 plus RU486.</p><p><b>CONCLUSION</b>Expression of IL-12 gene can be effectively controlled by a RU486 regulatory system. The inducible IL-12 can delay the growth of orthotopically transplanted hepatoma and prolong the survival of mice.</p>

Effects of 17β-estradiol on proliferation and migration of MCF-7 cell by regulating expression of claudin-6 / 中华病理学杂志

<p><b>OBJECTIVE</b>To explore the role of estrogen in the regulation of the expression of claudin-6 and biological behavior in MCF-7 cells.</p><p><b>METHODS</b>RT-PCR and immunocytochemistry were conducted to analyze the expression and localization of claudin-6 in MCF-7 cells treated with 17β-estradiol. CCK-8 kit assay and Scratch Test were conducted to analyze the capability of proliferation and migration of 17β-estradiol treated MCF-7 cells.</p><p><b>RESULTS</b>RT-PCR analysis and immunocytochemistry showed that 17β-estradiol induced a concentration-and time-dependent effect on claudin-6. At 5 nmol/L and at 24 h, 17β-estradiol treatment led to an increased level of claudin-6, which was located in the membrane of MCF-7 cells. CCK-8 analysis showed a significant decrease in the capability of proliferation of MCF-7 cells compared with the control group (P < 0.05). Cells Scratch Test showed decreased migration capability of MCF-7 cells compared with the control group (P > 0.05).</p><p><b>CONCLUSIONS</b>17β-E2 might regulate the expression of claudin-6 and inhibit the proliferation and migration of MCF-7 cells. The inhibitory effects of 17β-E2 on growth and migration of MCF-7 cells may be mediated by claudin-6 expression regulation.</p>

Inhibition of malignant phenotype of HeLa cells by RNA interference of the telomerase activity / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the biology of HeLa cells upon inhibition of human telomerase catalytic subunit (hTERT) gene by RNA interference in vitro.</p><p><b>METHODS</b>Four shRNAs (A, B, C and D) targeting hTERT gene were designed and prepared by in-vitro transcription. The expression of hTERT gene was evaluated by immunofluorescent staining and telomeric repeat amplification protocol (TRAP) ELISA (TRAP-ELISA), after transient transfection of shRNAs by lipid formulation. Through the initial selection, shRNA (B) was noticed as the most efficient one in down-regulating hTERT gene and therefore was chosen as the ultimate shRNA used in the experimental groups. Those transfected by non-silencing RNAi were chosen as the control groups. Cell spreading and migration were studied by microscopy and cell adhesion to fibronectin (FN) was assayed by cell counting kit-8 (CCK-8). Cell invasion was assessed by Boyden chamber assay.</p><p><b>RESULTS</b>Cell spreading study revealed that the rates of spreading cells in the experimental groups were (5.6 +/- 2.3)% at 30 min, and (26.3 +/- 6.1)% 2 h after the inoculation, respectively, whereas the rates of spreading cells in the control groups were (31.3 +/- 7.9)% and (79.4 +/- 4.8)%, respectively. There were significant differences between the two groups (P < 0.01). However, most of the cells in both groups became spreading after 24 h. Cell adhesion assay demonstrated that the rate of adhesion cells on FN in experimental groups was (67.2 +/- 2.8)%, less than that in control groups (83.7 +/- 5.4)% (P < 0.05). The relative migration distance was (27.1 +/- 6.2)% in the experimental group, lower than that of the control group (58.7 +/- 15.0)%. The invasion assay revealed that the invading cells were 75.7 +/- 14.5 in the experimental group, in contrast to 165.1 +/- 11.0 in the control group after 4 h incubation on matrigel. The difference between these two groups was significant (P < 0.05).</p><p><b>CONCLUSION</b>In vitro shRNA silencing of hTERT gene can down-regulate the telomerase activity, leading to an inhibition of the malignant phenotype of HeLa cells, including decreased ability of cell spreading and adhesion, reduction of cell migration, and declined invasive ability through Matrige assay.</p>

Effect of bone marrow mesenchymal stem cells on tumor neovascularization / 中华病理学杂志

<p><b>OBJECTIVE</b>The effect of human bone marrow mesenchymal stem cells (hMSCs) on tumor neovascularization were studied.</p><p><b>METHODS</b>hMSCs were isolated from human bone marrow by density gradient fractionation and adherence to plastic flasks. hMSCs-EGFP were obtained by pLEGFP-N1 retroviral vector. Flow cytometry was used to detect the cell surface antigen and the differentiation potential of hMSCs-EGFP was investigated under conditioned media. The effect of hMSCs on tumor neovascularization were observed by establishing solid tumor models in BALB/C nude mice. In addition, effect of the conditioned medium used for tumor cells and endothelial cells (EC) cultivation was collected, to detect its effect on the growth and migration rates of hMSC. hMSCs were induced to differentiate into EC in vitro and the migratory effect on HUVEC was also evaluated.</p><p><b>RESULTS</b>hMSCs-EGFP, like hMSC, exhibited a fibroblast-like morphological feature, and both had the similar cell surface antigens. They could be induced into osteocytes or adipocytes under the conditioned media. The results not only suggested that hMSCs contributed to tumor neovascularization, but also indicated that most of vessels were host-derived angiogenesis mediated by hMSCs. The mean vascular density (MVD) in suspension group (13.67 ± 1.53) was strikely higher than that in MCF-7 group (5.33 ± 1.42), which showed statistical significance (P < 0.05). Only very few vessels were attributed to hMSCs transdifferentiation into ECs. Tumor cells and ECs can promote hMSCs proliferation and migration through paracrine action. Furthermore, hMSCs were positive for CD31 after 2 weeks induction and HUVEC migration can be facilitated by hMSCs.</p><p><b>CONCLUSION</b>MSCs have the effect of promoting tumor neovascularization.</p>

Repair of calvarial defects with human umbilical cord blood derived mesenchymal stem cells and demineralized bone matrix in athymic rats / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of using human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) and demineralized bone matrix (DBM) scaffolds to repair critical-sized calvarial defects in athymic rats.</p><p><b>METHODS</b>Human UCB-MSCs were isolated, expanded and osteogenically induced in vitro. Osteogenic differentiation of UCB-MSCs was evaluated by Alizarin Red staining and measurement of calcium content respectively, and then the cells were seeded onto DBM scaffolds. Bilateral full-thickness defects (5 mm in diameter) of parietal bone were created in an athymic rat model. The defects were either repaired with UCB-MSC/DBM constructs (experimental group) or with DBM scaffolds alone (control group). Animals were harvested at 6 and 12 weeks post-implantation respectively, and defect repair was evaluated with gross observation, micro-CT measurement and histological analysis.</p><p><b>RESULTS</b>Micro-CT showed that new bone was formed in the experimental group at 6 weeks post-implantation, while no sign of new bone formation was observed in the control group. At 12 weeks post-transplantation, scaffolds had been degraded almost completely in both sides. It was shown that an average of (78.19 +/- 6.45)% of each defect volume had been repaired in experimental side; while in the control side, only limited bone formed at the periphery of the defect. Histological examination revealed that the defect was repaired by trabecular bone tissue in experimental side at 12 weeks, while only fibrous connection was observed in the control group.</p><p><b>CONCLUSIONS</b>Tissue-engineered bone composed of osteogenically-induced human UCB-MSCs on DBM scaffolds could successfully repair the critical-sized calvarial defects in athymic rat models.</p>

MT1-MMP up-regulates VEGF expression in human breast carcinoma MCF-7 cells and induces tumor angiogenesis / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the function of MT1-MMP in tumor angiogenesis and elucidate the possible way of action that MT1-MMP contributes to angiogenesis.</p><p><b>METHODS</b>MT1-MMP was transfected into human breast carcinoma cell line MCF-7 cells. Semi-quantitative RT-PCR and immunofluorescence staining were employed to detect the expression of VEGF in the transfected and non-transfected MCF-7 cells. Tumor growth, microvessel density and expression of VEGF in nude mice were detected through in vivo tumorigenicity assay.</p><p><b>RESULTS</b>In MT1-MMP stable transfected MCF-7 cells, mRNA expression of VEGF(189), VEGF(165), and VEGF(121) and immunofluorescence intensity were significantly elevated (P < 0.001). In vivo tumorigenicity assay in the nude mice showed that MT1-MMP promoted tumor growth. The MVD in the MT1-MMP-transfected cells-transplanted tumor tissue was significantly elevated (P < 0.05). Immunohistochemical assay showed that there was a strong immunostaining of VEGF in those tumor tissues.</p><p><b>CONCLUSION</b>MT1-MMP can induce tumor angiogenesis through up-regulation of VEGF expression. This function of MT1-MMP may open a new approach for clinical anti-tumor research and anti-tumor drug development.</p>

The cloning expression, purification and activity of human angiostatin K (1-3) gene / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To clone the sequence of human Angiostatin cDNA and obtain the protein of recombinant angiostatin for further development.</p><p><b>METHODS</b>Fresh human liver tissue was used for the extraction of total RNA and amplified the Angiostatin cDNA through RT-PCR method . After the recombinant plasmid pET30a-Angiostatin was constructed and confirmed, it was transduced into Rosetta (DE3). Then the transformation was used for fermentation and induced expression. The protein was identified by Western Blot and purified by Ni-NTA affinity chromatography.</p><p><b>RESULTS</b>The sequence of human Angiostatin cDNA was identical with genebank. Angiostatin (K1-3) was expressed and purified. The target protein made up 30% of the total bacterial protein. The purity is above 90%.</p><p><b>CONCLUSION</b>Angiostatin K (1-3) can be reached using fusion vector pET30a. The product have the biological activity.</p>

The dynamic expression of PTEN in fibrogenic rat liver tissues and its relation to the activation and proliferation of hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the dynamic expression of PTEN in fibrogenic liver tissue of rats and its effect on the activation and proliferation of hepatic stellate cells (HSC).</p><p><b>METHODS</b>A rat model of hepatic fibrosis was established by common bile duct ligation (BDL). The expressions of PTEN in the rat liver tissues were detected by immunohistochemical staining, Western blot and real-time PCR assay. The expressions of PTEN in activated HSC in the rat liver tissues were detected by immunofluorescence double labeling confocal laser scanning microscopy. The alpha-SMA in the rat liver tissues was determined by immunohistochemical staining.</p><p><b>RESULTS</b>The immunohistochemical staining indicated that there was extensive expression of PTEN in the liver tissues of normal rats, it was expressed mainly in the cytoplasm of the HSC. With the aggravation of hepatic fibrosis, the expression of PTEN in the hepatic tissues decreased gradually (P less than 0.01), while the alpha-SMA positive cells in the hepatic tissues increased significantly (P less than 0.01). The expressions of PTEN protein and mRNA in the rat liver tissues at week 1, 2, 3 and 4 after BDL were all lower than those in the sham operation group (P less than 0.01), and the expressions gradually decreased with the development of hepatic fibrosis (P less than 0.01). Immunofluorescence double labeling confocal laser scanning microscopy showed that PTEN were expressed extensively in activated HSC, especially in the cytoplasm, and with the development of hepatic fibrosis, the PTEN-expressing activated HSC accounted for an increasingly smaller percentage of total activated HSC.</p><p><b>CONCLUSION</b>The expressions of PTEN mRNA and protein in rat fibrogenic liver tissues were downregulated, and their expressions in HSC in vivo also decreased. The dynamic expressions of PTEN in liver tissues had a significant negative correlation with the activation and proliferation of HSC.</p>