Comparison of coagulation function between adrenocorticotropic hormone independent Cushing syndrome and nonfunctional adrenal adenoma and its influence factors / 北京大学学报(医学版)

OBJECTIVE@#To investigate the coagulation function indicators and identify influence factors of hypercoagulability in patients with adrenocorticotropic hormone (ACTH) independent Cushing syndrome (CS).@*METHODS@#In our retrospective study, the electronic medical records system of Peking University First Hospital was searched for the patients diagnosed with ACTH independent CS on discharge from January 2014 to June 2019. Nonfunctional adrenal adenoma patients were chosen as control group and matched 1 ∶1 by body mass index (BMI), gender, and discharge date. Clinical features and coagulation function indicators were compared between the two groups.@*RESULTS@#In the study, 171 patients were included in each group. Compared with control group, activated partial thromboplastin time (APTT), and prothrombin time (PT) in ACTH independent CS group were significantly lower [(29.22±3.39) s vs. (31.86±3.63) s, P < 0.001; (29.22±3.39) s vs. (31.86±3.63) s, P < 0.001], and both D-dimer and fibrin degradation products (FDP) levels were significantly higher (P < 0.05). Percentage of APTT levels under the lower limit of reference range in the CS patients was significantly higher than that in nonfunctional group (21.6% vs. 3.5%, P < 0.001). Percentage of D-dimer levels over the upper limit of reference range in the CS patients was significantly higher than that in nonfunctional group (13.5% vs. 6.6%, P=0.041). There were three patients with deep venous thrombosis and one patient with pulmonary embolism in CS group, however none was in control group. The area under curve (AUC) of serum cortisol rhythm (8:00, 16:00 and 24:00) levels was negatively associated with the levels of PT (r=-0.315, P < 0.001) and APTT (r=-0.410, P < 0.001), and positively associated with FDP (r=0.303, P < 0.001) and D-dimer levels (r=0.258, P < 0.001). There were no differences in coagulation function indicators among different histopathologic subgroups (adrenocortical adenoma, adrenocortical hyperplasia, oncocytic adenoma, adrenocortical carcinoma). With Logistic regression analysis, the AUC of cortisol and glycosylated hemoglobin A1c (HbA1c) levels were independent risk factors for hypercoagulability in the ACTH independent CS patients (P < 0.05).@*CONCLUSION@#ACTH independent CS patients were more likely in hypercoagulable state compared with nonfunctional adrenal adenoma, especially in ACTH independent CS patients with higher levels of cortisol AUC and HbA1c. These patients should be paid attention to for the hypercoagulability and thrombosis risk.

Preliminary observation on the clinical application of rehabilitation support for lumbar disc herniation based on 3D printing technology / 中国骨伤

OBJECTIVE@#To analyze the important effect of 3D printing personalized lumbar support on lumbar pain and lumbar function in patients with lumbar disc herniation.@*METHODS@#From October 2018 to May 2021, 60 patients initially diagnosed with lumbar disc herniation were selected and divided into an observation group and a control group, with 30 patients in each group. Among them, there were 18 males and 12 females in the observation group;the age ranged from 24 to 56 years old, with an average of (45.23±6.07) years old. The course of disease ranged from 1 to 24 months, with an average of(6.25±0.82) months, and rehabilitation treatment was carried out by wearing 3D printed personalized lumbar support. There were 19 males and 11 females in the control group;the age ranged from 25 to 57 years old, with an average of (42.78±7.58) years old. The course of disease ranged from 1 to 24 months, with an average of (6.72±1.36) months, and rehabilitation treatment is carried out by wearing traditional lumbar protective equipment. The Japanese Orthopaedic Association (JOA) scores, lumbar Oswestry dysfunction index (ODI) and visual analogue scale (VAS) were evaluated and compared between the two groups before and 1 course after treatment (3 weeks).@*RESULTS@#There was no statistically significant difference in JOA, ODI, and VAS between two groups before treatment (P>0.05). After one course of treatment (3 weeks), JOA scores of both groups was increased compared to before treatment (P<0.05), while ODI and VAS decreased compared to before treatment (P<0.05). After treatment, JOA score of observation group was higher than that of control group (P<0.05), while ODI and VAS scores were lower than those of control group. No adverse events occurred in both groups.@*CONCLUSION@#The application of 3D printing personalized lumbar support can effectively alleviate the pain of patients with lumbar disc herniation and improve their lumbar function of patients.

Baicalin enhances middle cerebral artery smooth muscle cells BK channel-mediated outward currents in SD rats / 中国药理学通报

Aim To study the effeet of baicalin on middle cerebral arterv in SD rats.Methods The j changes of middle cerebral artery diameter were observed using a pressure myograph system.The whole- cell and inside-out patch-clamp recording were used to detected the electrophysiological features of single vascular smooth muscle cells.Results Baicalin dilated the middle cerebral artery segment of SD rats in a con- centration-dependent manner.IbTX blocked baicalin - mediated relaxation.Baicalin enhanced the outward current of middle cerebral artery smooth muscle cells in a concentration-dependent manner.IbTX blocked ba- icalin-mediated outward current.Baicalin increased BK channel open probabilities.Conclusions Baicalin enhances the outward current mediated by BK channel and relaxes the middle cerebral arterv in SD rats.

Analyzing the influencing factors of work-related musculoskeletal disorders among manufacturing painters / 中国职业医学

@#Objective - To analyze the prevalence and influencing factors of work related musculoskeletal disorders (WMSDs) Methods among painters in the manufacturing industry. A total of 639 painters from one shipbuilding enterprise, one automobile manufacturing enterprise and three wooden furniture manufacturing enterprises in Guangdong Province were selected as the research subjects using typical sampling method. The Chinese version of Musculoskeletal Disorders Questionnaire was Results used to investigate the prevalence of WMSDs in the past one year, and the influencing factors were analyzed. The total prevalence rate of WMSDs among painters in the manufacturing industry was 37.4%. The prevalence of WMSDs in different vs vs P industries from high to low was shipbuilding, automobile and furniture manufacturing (50.0% 38.7% 29.0%, <0.01). The prevalence of WMSDs in different parts of the body from high to low was neck, ankle/foot, shoulder, low back, upper back, knee, vs vs vs vs vs vs vs vs P hand/wrist, hip/leg and elbow (20.7% 19.2% 17.4% 15.8% 14.1% 13.8% 13.5% 9.5% 6.6%, <0.01). Multivariate logistic regression analysis results showed that working in uncomfortable postures was a risk factor for neck, ankle/ P P foot and shoulder WMSDs (all <0.01); long time head turning was a risk factor for neck and shoulder WMSDs (both <0.05); P overweight and obesity, and bending and turning frequently at the same time were risk factors for ankle/foot WMSDs (all <0.05); P adequate rest time was a protective factor for neck and ankle/foot WMSDs (both <0.01); participated in physical exercise more P than once a week was a protective factor of neck and shoulder WMSDs in painters (all <0.05), after excluding the influence of Conclusion confounding factors. The prevalence of WMSDs in manufacturing painters was high, and the main body parts E mail 4813545@qq.com E mail wangzhongxu2003@163.com· · 中国职业医学 年 月第 卷第 期 ， ， ， 482 2022 10 49 5 Chin Occup Med October 2022 Vol.49 No.5 involved were neck, ankle/foot and shoulder. The influencing factors include individual factors, poor ergonomics factors and unreasonable work organization.

Curcumin targets vascular endothelial growth factor viaactivating the PI3K/Akt signaling pathway and improves brainhypoxic-ischemic injury in neonatal rats

This study aimed to evaluate the effect of curcumin on brain hypoxicischemic(HI) damage in neonatal rats and whether the phosphoinositide 3-kinase(PI3K)/Akt/vascular endothelial growth factor (VEGF) signaling pathway is involved.Brain HI damage models were established in neonatal rats, which received the followingtreatments: curcumin by intraperitoneal injection before injury, insulin-likegrowth factor 1 (IGF-1) by subcutaneous injection after injury, and VEGF by intracerebroventricularinjection after injury. This was followed by neurological evaluation,hemodynamic measurements, histopathological assessment, TUNEL assay,flow cytometry, and western blotting to assess the expression of p-PI3K, PI3K, p-Akt,Akt, and VEGF. Compared with rats that underwent sham operation, rats with brainHI damage showed remarkably increased neurological deficits, reduced right bloodflow volume, elevated blood viscosity and haematocrit, and aggravated cell damageand apoptosis; these injuries were significantly improved by curcumin pretreatment.Meanwhile, brain HI damage induced the overexpression of p-PI3K, p-Akt, and VEGF,while curcumin pretreatment inhibited the expression of these proteins. In addition,IGF-1 treatment rescued the curcumin-induced down-regulated expression of p-PI3K, p-Akt, and VEGF, and VEGF overexpression counteracted the inhibitory effectof curcumin on brain HI damage. Overall, pretreatment with curcumin protectedagainst brain HI damage by targeting VEGF via the PI3K/Akt signaling pathway inneonatal rats.

Curcumin targets vascular endothelial growth factor viaactivating the PI3K/Akt signaling pathway and improves brainhypoxic-ischemic injury in neonatal rats

This study aimed to evaluate the effect of curcumin on brain hypoxicischemic(HI) damage in neonatal rats and whether the phosphoinositide 3-kinase(PI3K)/Akt/vascular endothelial growth factor (VEGF) signaling pathway is involved.Brain HI damage models were established in neonatal rats, which received the followingtreatments: curcumin by intraperitoneal injection before injury, insulin-likegrowth factor 1 (IGF-1) by subcutaneous injection after injury, and VEGF by intracerebroventricularinjection after injury. This was followed by neurological evaluation,hemodynamic measurements, histopathological assessment, TUNEL assay,flow cytometry, and western blotting to assess the expression of p-PI3K, PI3K, p-Akt,Akt, and VEGF. Compared with rats that underwent sham operation, rats with brainHI damage showed remarkably increased neurological deficits, reduced right bloodflow volume, elevated blood viscosity and haematocrit, and aggravated cell damageand apoptosis; these injuries were significantly improved by curcumin pretreatment.Meanwhile, brain HI damage induced the overexpression of p-PI3K, p-Akt, and VEGF,while curcumin pretreatment inhibited the expression of these proteins. In addition,IGF-1 treatment rescued the curcumin-induced down-regulated expression of p-PI3K, p-Akt, and VEGF, and VEGF overexpression counteracted the inhibitory effectof curcumin on brain HI damage. Overall, pretreatment with curcumin protectedagainst brain HI damage by targeting VEGF via the PI3K/Akt signaling pathway inneonatal rats.

Clinical characteristics of 4355 patients with anterior cruciate ligament injury / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Clinical features of anterior cruciate ligament (ACL) injury are important for its prevention, diagnosis and treatment. However, few studies have reported such data, especially in China. The purpose of this study was to describe the clinical characteristics of ACL injury on a large cohort.</p><p><b>METHODS</b>Between 1993 and 2007, a total of 4355 ACL deficient inpatients (612 athletes and 3743 non-athletes) were registered. Data were collected using a special database system. And the distributions of characteristics in different groups were compared and analyzed statistically.</p><p><b>RESULTS</b>All subjects were confirmed with ACL tear during surgery. Statistical analysis revealed that the percentage of females in Athlete Group was significantly higher than that in Non-athlete Group (56.05% vs. 24.95%, P < 0.001). This study also found that sports trauma was the main cause of ACL tears. Soccer, basketball, judo, wrestling and track and field were the five most responsible activities for athletes. The average injury time for athletes was significantly shorter than that for non-athletes (413.3 days vs. 717.5 days, P < 0.001). Three thousand nine hundred and eight cases were ordered ACL reconstruction (76.04% single-bundle, 18.30% double-bundle). Three hundred and forty-five patients (7.92%) were combined with other ligaments injuries, 2667 (61.24%) were found with various grades of cartilage lesions, and 3377 (77.54%) were found with meniscal injury.</p><p><b>CONCLUSIONS</b>Sports trauma was the main cause of ACL tears in China, and reconstruction had become the principal surgical choice. In order to restore knee joint stability and reduce the incidence of cartilage and meniscal injury, patienttailored ACL reconstruction should be suggested at the right moment.</p>

Effect of conditioned medium of mesenchymal stem cells on proliferation, migration and adhesion of human umbilical vein endothelial cells / 中国实验血液学杂志

The aim of this study was to explore the effect of mesenchymal stem cell (MSC) conditioned medium (MSC-CM) on proliferation, migration and adhesion of human umbilical vein endothelial cell (CRL1730) and its mechanism. Isolation and purification of MSC were performed with the classic adhering method, the surface markers (CD29, CD90, CD45 and CD34) in MSC were detected by flow cytometry. MSC were treated and cultured for 3 d, the MSC-CM or MSC overexpressing stem cell-derived factor-1 (SDF-1) conditioned medium (Ad-SDF-1-MSC-CM) were collected. Subsequently, CRL1730 cells were treated respectively with 2% FBS-DMEM, 15% FBS-DMEM (control group), MSC-CM or Ad-SDF-1-MSC-CM for 24 h, the proliferation of CRL1730 cells was detected by MTT method. CRL1730 cell migration in vitro was performed by using wound healing system. The adhesion ability of CRL1730 cells was analyzed by microscope. The results indicated that the CRL1730 cells treated with Ad-SDF-1-MSC-CM showed greater proliferative capacity than CRL1730 cells treated with MSC-CM. While adding with AMD3100 5 µmol/L, the blocker of CXCR4, the CRL1730 proliferation mediated by Ad-SDF-1-MSC-CM was significantly reduced. Meanwhile, compared with MSC-CM, Ad-SDF-1-MSC-CM had greater effects for promoting CRL1730 migration and enhancing adhesion ability of CRL1730 cells, these effects were significantly inhibited by AMD3100. It is concluded that MSC-CM promotes the migration and adhesion ability of CRL1730 cells through SDF-1 expressed by MSC.

VEGF promotes the proliferation of bone marrow derived mesenchymal stem cells through ERK1/2 signal pathway / 中国实验血液学杂志

In order to explore the effect of VEGF on mesenchymal stem cell (MSC) proliferation and its possible signal transduction mechanism, MSC culture was performed with the classical bone marrow adhering method; characteristics of passage 3 rat MSC (P3MSC) was identified through multi-differentiation and surface marker assay (CD34, CD45, CD90, CD29); P3MSC were treated with 20 ng/ml VEGF, and the effect of VEGF on the MSC proliferation was measured during 12, 36 and 72 hours by MTT assay. Subsequently, P3MSC were treated with extracellular-signal regulated kinase (ERK1/2) inhibitor PD98059 (50 µmol/L) or p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580 (30 µmol/L) for 30 minutes, the culture medium was replaced with new medium including 20 ng/ml VEGF. After 72 hours, the effect of PD98059 or SB203580 on MSC proliferation mediated by VEGF was measured by MTT assay. The result showed that the cultured MSC expressed PDGFR-α, PDGFR-β and NRP1, but did not express VEGF-R (Flk1 and Flt1). The MSC had the multi-differentiation ability and displayed the characteristics of CD90+ (96.7%), CD29+ (94.6%), CD34- (0.79%) and CD45- (0.84%). The MSC proliferation rate increased gradually with prolonging of the functioning time of 20 ng/ml VEGF, and MSC proliferation rate may reach to maximum value after treating with 20 ng/ml VEGF for 72 hours. The effect of VEGF on MSC proliferation was found to be abolished, even was under level of control group after treating with PD98059 or SB203580 for 30 minutes. Furthermore, the inhibitory effect of PD98059 on MSC proliferation was obviously higher than that of SB203580. It is concluded that the VEGF can promote MSC proliferation, and its possible mechanism may relate to ERK1/2 pathway.

Effect of mesenchymal stem cells transfected with human vegf-165 gene carried by adenovirus on revascularization for hind limb ischemic necrosis in rat model / 中国实验血液学杂志

The aim of study was to explore the effect of mesenchymal stem cells (MSC) transfected with recombinant adenovirus-mediated human vascular endothelium growth factor 165 (ad-vegf-165) on treating ischemic necrosis limbs. Adult SD rats were selected for study. Limb ischemic necrosis model was established by right femoral artery ligation in SD rats. 7 days after ligation, MSC, ad-h-vegf-165-MSC and ad-LacZ-MSC labelled by DAPI were injected into ischemic necrosis limb in rats respectively. One week after injection, the expression of VEGF in ischemic necrosis limbs was detected by Western blot. And at 1, 2 or 4 weeks after injection, the expressions of FVIII and myosin on MSC were evaluated by immunohistochemistry. The results indicated that the MSC labelled by DAPI could be found in the transplantation site of ischemic necrosis limbs under fluorescent microscope. And the number of MSC in MSC-vegf group was more than that in MSC and MSC-LacZ groups. The VEGF expression in MSC-vegf group was higher than that in MSC and MSC-LacZ groups. More importantly, the number of endothelial cells demonstrated characteristic FVIII positive MSC in MSC-vegf group was more than that in MSC and MSC-LacZ group after injections of 1, 2 and 4 weeks. However, the number of myosin positive MSC among MSC-vegf, MSC and MSC-LacZ groups showed no significant difference. It is concluded that MSC transfected with Ad-vegf promotes angiogenesis to repair ischemic necrosis limbs through the increased expression of VEGF.

Effect of adenovirus-mediated stromal cell-derived factor-alpha gene transfer on ventricular remodeling in rats with myocardial infarction / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the effect of adenovirus-mediated human stromal cell-derived factor-1alpha (hSDF-1alpha) on ventricular remodeling in rats with myocardial infarction.</p><p><b>METHODS</b>A recombinant adenoviral plasmid containing hSDF-1alpha cDNA was constructed using homologous recombination in bacteria and the recombinant adenovirus particles expressing hSDF-1alpha (AdV-SDF-1) were prepared. In rat models of myocardial infarction induced by left anterior descending artery occlusion, 1x10(10) PFU AdV-SDF-1 or PFU AdV-LacZ were injected at multiple sites into the infarcted myocardium 1 h after the operation, using 200 l cell-free PBS as the control. Four weeks after the injection, the cardiac function of the rats was analyzed, and the heart tissues were taken after the measurement of hemodynamics. On serial frozen sections, histological observation and morphometric measurement were carried out using a microscopic image analysis system, and the expression of hSDF-1alpha was detected by immunocytochemistry.</p><p><b>RESULTS</b>Four weeks after AdV-SDF-1 injection, the myocardium in the infracted area showed significantly higher expression rates of hSDF-1alpha. The injection resulted in a obvious reduction in the infarct size and collagen content and a marked increase in the left ventricle wall, and the rats showed improved cardiac functions.</p><p><b>CONCLUSION</b>SDF-1alpha can improve the cardiac structure and function in rats with myocardial infarction by inhibiting collagen synthesis and deposition in the infarcted area.</p>

Protective effect of preconditioning with PEP-1-CAT fusion protein against myocardial ischemia-reperfusion injury in rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the transduction efficiency of purified PEP-1-CAT fusion protein into rat heart and the protective effect of the fusion protein against myocardial ischemia-reperfusion injury.</p><p><b>METHODS</b>PEP-1-CAT or CAT (500 microg) was injected in SD rats via the caudal vein, using normal saline as the control, and the hearts were harvested at 0.5, 1, 2, 4, 8, and 24 h after the injection. The transduction efficiency was evaluated by immunofluorescence technique, and the CAT activity was measured. Forty rats were randomized into 5 groups, namely the sham-operated group, ischemia-reperfusion group, and 3 PEP-1-CAT -treated groups (100, 300, and 500 microg). The left main coronary artery was occluded for 1 h followed by a 2-h reperfusion, and at the end of reperfusion, serum LDH and CK and MDA content in the myocardium were measured.</p><p><b>RESULTS</b>No green fluorescence was observed in saline group or CAT group. Bright green fluorescence was observed in PEP-1-CAT groups at different time points, most conspicuous at 8 h. No significant difference in CAT activity was found between CAT group and saline group (P>0.05); with the lapse of time, CAT activity in PEP-1-CAT group increased gradually, reaching the peak level at 8 h, which was 4.2 folds of that in the saline group. LDH ,CK and MDA were significantly lower in PEP-1-CAT- groups than in ischemia-reperfusion group (P<0.01).</p><p><b>CONCLUSION</b>PEP-1 can mediate the transduction of CAT in rat heart in a time-dependent manner, and PEP-1-CAT preconditioning provides a protective effect against ischemia- reperfusion injury in rats.</p>

Mechanism of granulocyte colony-stimulating factor for promoting cell viability of bone marrow mesenchymal stem cells / 生理学报

The present study was aimed to investigate the mechanism of the granulocyte colony-stimulating factor (G-CSF) on the viability of the bone marrow mesenchymal stem cells (MSCs). MSCs were cultured by classical whole bone marrow adhering method, and the MSCs were analyzed for the cell surface differentiation markers CD34, CD133, CD90 and CD105 by flow cytometry (FCM). The ability of the MSCs to differentiate into osteocytes and adipocytes was tested in osteogenic and adipogenic mediums, separately. The effect of G-CSF (20 mug/mL) on the passage 3 MSCs viability was evaluated by MTT method, and the molecular mechanism of the G-CSF mediated effects was assayed through the pretreatment of the signal pathway inhibitors including 50 nmol/L wortmannin (phosphatidylinoesitol 3 kinase inhibitor), 50 mumol/L PD98059 [extracellular signal-regulated-kinase1/2 (ERK1/2) inhibitor], 30 mumol/L SB203580 (p38 mitogen-activated protein kinase inhibitor), 10 mumol/L H89 (protein kinase A inhibitor), 20 mumol/L Y27632 (Rho kinase inhibitor), 1 mumol/L rapamycin [mammalian target of rapamycin (mTOR) inhibitor], 10 mmol/L straurosporine [protein kinase C (PKC) inhibitor], 6 nmol/L G0697 (PKCalpha inhibitor) and 50 mumol/L Pseudo Z (PKCzeta inhibitor). Cultured passage 3 MSCs expressed CD90 and CD105 strongly, and showed the ability of multi-differentiation into osteocytes and adipocytes. G-CSF promoted the viability of MSCs, and the promotion was completely inhibited by PKC inhibitor straurosporine and partially inhibited by wortmannin, rapamycin, PD98059, SB203580 or G0697. However, its effect was not inhibited by H89, Y27632 and Pseudo Z. It is thus suggested that the promoting effect of G-CSF on MSCs viability was closely related to AKT-mTOR-PKC signal pathway, and PKC maybe the central role in the signal pathway.

Comparison of migration characteristics of MSCs in different assay systems / 中国实验血液学杂志

The aim of this study was to explore the difference of MSC migration mediated by SDF-1/CXCR4 axis through Boyden chamber in vitro migration assay. The SDF-1 density-dependence of MSC migration was observed. Subsequently, the effects of different blocking agents on hSDF-MSC migration were observed after MSC were treated with 50 nmol/L wortmannin, 10 micromol/L LY294002, 50 micromol/L PD98059, 10 micromol/L U73122, 126 micromol/L AMD3100 and 50 nmol/L verapamil respectively. The results showed the efficiency of MSC migration increased gradually with the increasing of hSDF-1 density. And after MSCs treatment with 50 nmol/L wortmannin, 10 micromol/L LY294002, 50 micromol/L PD98059, 10 micromol/L U73122 and 126 micromol/L AMD3100 respectively, the ability of MSC migration decreased. The ability of MSCs migration obviously decreased when MSCs were treated with U73122, AMD3100. It is concluded that the SDF-1/CXCR4-mediated MSC migration may be related to mitogen-activated protein kinase (MAPK), phosphatidylinositol phospholipase C (PI-PLC) and protein kinase (PKC) signal pathways.

The role of PEP-1-SOD1 fusion protein on ischemia-reperfusion injury in isolated perfused rat hearts / 中华心血管病杂志

<p><b>OBJECTIVE</b>The transduction efficiency of the purified PEP-1-SOD1 fusion protein and the effects of PEP-1-SOD1 fusion protein on ischemia reperfusion injury in the isolated perfused rat hearts were investigated.</p><p><b>METHODS</b>The constructed pET15b-SOD1 and pET15b-PEP-1-SOD1 were transformed into BL21 (DE3) for expression and purification of SOD1 and PEP-1-SOD1, respectively. Isolated perfused rat hearts were subjected to 60 min of global ischemia and 30 min of reperfusion and treated with vehicle, 100 micromol/L SOD1 and 25, 50, 100 micromol/L PEP-1-SOD1, respectively. The transduction efficiency was evaluated with immunofluorescent microscopy and Western blot. The enzyme activity of the transduced PEP-1-SOD1 was measured with commercial SOD detection kit. The MDA content in myocardial tissue and the CK activity in coronary exudate at 15 min after reperfusion were also measured. Cardiomyocyte apoptosis was detected with TUNEL. The infarct size was determined in isolated hearts 60 min after reperfusion with TTC staining.</p><p><b>RESULTS</b>Immunofluorescent microscopy and Western blot demonstrated PEP-1-SOD1 was transduced into myocardial tissue in a dose-dependent manner, whereas SOD1 could not be detected in SOD1 group. SOD activity in control, SOD1 group, 25, 50, 100 micromol/L PEP-1-SOD1 groups was (10.06 +/- 0.77) U/mg prot, (10.59 +/- 0.71) U/mg prot, (32.29 +/- 1.42) U/mg prot, (43.16 +/- 1.16) U/mg prot, (55.14 +/- 1.59) U/mg prot, respectively. MDA content in corresponding groups was (1.48 +/- 0.19) nmol/mg prot, (1.39 +/- 0.11) nmol/mg prot, (1.01 +/- 0.14) nmol/mg prot, (0.73 +/- 0.13) nmol/mg prot, (0.50 +/- 0.06) nmol/mg prot, respectively. CK activity in corresponding groups was (1.73 +/- 0.58) U/mg prot,(1.68 +/- 0.14) U/mg prot,(1.40 +/- 0.28) U/mg prot,(0.97 +/- 0.39) U/mg prot, (0.61 +/- 0.56) U/mg prot, respectively. Cardiomyocyte apoptotic index in corresponding groups was (17.25 +/- 0.75)%, (16.63 +/- 1.07)%, (11.50 +/- 0.57) U/mg prot, (6.50 +/- 0.63) U/mg prot, (4.13 +/- 0.52)%, repectively. The percentage of myocardial infarction area was (55.13 +/- 2.18)%, (52.13 +/- 2.59)%, (33.88 +/- 2.06)%, (25.50 +/- 2.16)%, (15.38 +/- 1.14)%, respectively. Compared with control group and SOD1 group, all P < 0.01 These results demonstrated the enzyme activity of the transduced PEP-1-SOD1 was significantly increased in a dose-dependent manner and the MDA content, CK activity, the cardiomyocyte apoptotic index and the infarct size was decreased siginificantly in PEP-1-SOD1 pretreatment groups compared with SOD1 group.</p><p><b>CONCLUSION</b>The native, biologically active form of PEP-SOD1 fusion protein could be effectively transduced into the isolated rat hearts subjecting ischemia reperfusion injury in a dose-dependent manner. The transduced PEP-1-SOD1 has protective effects on ischemia reperfusion injury in the isolated rat hearts.</p>

Effects of different doses of thrombopoietin on proliferation of bone marrow mesenchymal stem cells in mice / 中国实验血液学杂志

To explore the effect of different doses of thrombopoietin on proliferation of bone marrow mesenchymal stem cells (MSCs) in mice, 20 Kunming mice (35 +/- 5 g) were divided randomly into 4 groups: low-dose TPO group, moderate-dose TPO group, high-dose TPO group and normal control group (n = 5). The experimental groups were subjected to intraperitoneal injections of TPO at a dose of 25, 50, 100 microg/kg, respectively, and normal control group were treated with saline at a dose of 0.1 ml/g per day for 5 days. The bone marrow was harvested on 12 hours after the final administration. The bone marrow nucleated cells (BMNCs) were counted and seeded at a density of 10(6) cells/cm(2). The colony-forming unit-fibroblast (CFU-F) of MSCs was cultured and evaluated. The CFU-F of MSCs underwent osteo-genic induction and adipogenic induction, and cytochemical and immunocytochemical staining were performed to verify their multipotential. CFU-F and the cell percentage of CD90(+), CD105(+), CD34(+) in BMNCs were analyzed by flow cytometry. The results showed that the number of BMNCs and the cell percentage of CD90(+), CD105(+), CD34(+) and CFU-F increased obviously in TPO groups as compared with the normal control group (p < 0.05). The number of BMNCs increased most obviously in the 50 microg/kg TPO group. However, there was no significant difference in number of CFU-F between 50 microg/kg and 100 microg/kg TPO group (p > 0.05). The CFU-F of MSCs in bone marrow had their osteogenic and adipogenic differentiation potentials in vitro. It is concluded that the number of BMNCs and the cell percentage of CD90(+), CD105(+) and CFU-F increased after administration with TPO. It means that TPO can enhance MSCs to proliferate in bone marrow. However, the number of BMNCs and CFU-F can not increase with the increase of TPO dose.

Adenovirus-mediated stromal cell-derived factor-1 alpha gene transfer promotes mesenchymal stem cell migration / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of stromal-derived factor-1 (SDF-1) in the migration of mesenchymal stem cells (MSCs) and the underlying signal transduction mechanism.</p><p><b>METHODS</b>Rat bone marrow-derived MSCs were infected with 100 ml recombinant adenovirus containing human SDF-1alpha gene (Ad-hSDF-1alpha), and the cell migration changes were observed at 1, 2, and 3 days after the infection. Twelve hours after Ad-hSDF-1alpha transfection, the MSCs in separate cultures were treated with wortmannin (50 nmol/L), LY294002 (10 mmol/L), PD98059 (50 mmol/L), U73122 (10 mmol/L), AMD3100 (0.1 g/L), or verapamil (50 nmol/L), respectively, and the signal transduction pathways involved in MSC migration were analyzed.</p><p><b>RESULTS</b>The MSCs grew in colonies after transfection with Ad-hSDF-1alpha, but this growth pattern was substantially attenuated after treatment with wortmannin, LY294002, PD98059, U73122, AMD3100 and verapamil, among which U73122 and AMD3100 treatments resulted in the most conspicuous inhibitory effects.</p><p><b>CONCLUSION</b>The effect of SDF-1 in promoting MSC migration is related to mitogen-activated protein kinase, phosphatidylinositol phospholipase C, and protein kinase signal pathways.</p>

Erythropoietin promotes proliferation of human bone marrow mesenchymal stem cells in vitro / 中国实验血液学杂志

This study was aimed to investigate the effects of recombinant human erythropoietin (rhEPO) on proliferation of human bone marrow-derived mesenchymal stem cells (MSCs) in vitro. The aspirates of the bone marrow from healty volunteers were seeded in culture medium. Then MSCs were isolated according to characteristics adhering to the plastics. After three passages in culture, bone marrow-derived adherent cells were identified by growing morphological features, cell surface antigens and differentiation into multi-lineages. Then P3-MSCs which had been identified were incubated with different concentrations of rhEPO (0.5, 1, 5, 10 and 50 U/ml). Subsequently, proliferation of MSCs was measured by MTT assay, as well as cell counts. At the same time, cell cycle was detected by flow cytometry (FCM). The results indicated that the expressions of CD90 and CD105 in P3 bone marrow-derived adherent cells were positive, while the expressions of CD34 and CD45 were negative, and these cells could differentiate into adipocytes, osteocytes and chondrocytes in induction media. MTT assay showed that the optical density (OD) of group treated with EPO was significantly higher than that in the control group (p<0.05), and the group treated with 50 U/ml EPO achieved the most predominant effects. The results of cell count were coincident with that of MTT assay. Furthermore, the cell cycle analysis by FCM revealed that rhEPO could relatively decrease the cell ratio in G0/G1 phase, and increase the cell ratio in S and G2/M phases. As compared with the control group, all those differences were statistically significant (p<0.01). It is concluded that erythropoietin can promote proliferation of human bone marrow mesenchymal stem cells in vitro, which may be correlated with the increased entry into S and M phases of cell cycle of MSCs adjusted by EPO.

The protective effect of PEP-1-SOD1 preconditioning on hypoxia/reoxygenation injury in cultured human umbilical vein endothelial cells / 中华心血管病杂志

<p><b>OBJECTIVE</b>To construct prokaryotic expression vector of pET15b-PEP-1-SOD1 and investigate whether PEP-1-SOD1 fusion protein could be transduced into human umbilical vein endothelial cells (HUVECs) and the effects on hypoxia/reoxygenation injury.</p><p><b>METHODS</b>The recombinant plasmids pET15b-SOD1 and pET15b-PEP-1-SOD1 were constructed and transformed into E. coli BL21 (DE3) to express SOD1 and PEP-1-SOD1 with an N-terminal His-tag. The purified SOD1 and PEP-1-SOD1 were incubated with HUVECs and the viability (MTT assay) and the release of lactate dehydrogenase (LDH) in culture medium were determined in the hypoxia/reoxygenation injury model. The morphological changes were observed under an inverted phase contrast microscope. The content of malondialdehyde (MDA) in HUVECs was also determined with the method of thiobarbituric acid.</p><p><b>RESULTS</b>PEP-1-SOD1 fusion protein could be transduced into cultured HUVECs in a time- and dose-dependent manner. The intracellular enzymatic activity of PEP-1-SOD1 after 30 min incubation with HUVECs was significantly higher than control group (60.88 U/ml +/- 6.73 U/ml vs. 41.06 U/ml +/- 4.19 U/ml, P < 0.01). The transduced PEP-1-SOD1 protein was enzymatically stable for 24 h within cells. After hypoxia/reoxygenation injury, control HUVECs shrunk, became round-shaped and intercellular space increased, while these morphological changes were not observed in PEP-1-SOD1 transduced HUVECs. PEP-1-SOD1 transduction also markedly increased the viability, decreased LDH leakage into culture media and reduced the content of MDA post hypoxia/reoxygenation.</p><p><b>CONCLUSIONS</b>PEP-1-SOD1 fusion protein could be efficiently transduced into HUVECs in a natively active form, and the delivered enzymatically active PEP-1-SOD1 exhibits cellular protection against hypoxia/reoxygenation injury in HUVECs. The transduction of SOD1 mediated by cell-penetrating peptide, PEP-1, provides a basis for further research on the prevention of ischemia/reperfusion injury in vivo.</p>

Effects of rhG-CSF on mobilization of mouse mesenchymal stem cells / 中国实验血液学杂志

To evaluate the effects of rhG-CSF on mobilization of mesenchymal stem cells (MSCs) of mouse bone marrow at different time point, thirty mice were randomly divided into rhG-CSF treatment group and control group. The mice were subcutaneously injected with rhG-CSF in a dose of 80 microg/kg or saline for 5 days. The bone marrow and peripheral blood were obtained at time points of 6, 12, 168 hours after final injection of rhG-CSF or saline. Bone marrow mononuclear cells (BMMNCs) were seeded at density of 1 x 10(6) MNCs onto 12-well plate for culture expansion in DMEM supplemented with 10% FBS, and the number of colony forming unit - fibroblast (CFU-F) was counted after 14 days. The cells were collected by trypsinization and the surface antigens CD34, CD133, CD90 and CD105 were analyzed by flow cytometry. The multi-differentiation of MSCs were done in the culture condition of induced-adipocyte and osteocyte. Peripheral blood MNCs examination was same as the bone marrow. The results indicated that the number of CFU-F of bone marrow in rhG-CSF group was more than that in control group (p < 0.01), the number of CFU-F in rhG-CSF group at 6 hours was more than that at 12 hours and 168 hours, respectively (p < 0.01). There was no obvious difference between CFU-F at 12 hours and at 168 hours (p > 0.05). MSCs were positive for CD90, CD105 and negative for CD34 and CD133. MSCs were found to differentiate into adipocyte and osteocyte in vitro. The CFU-F of PBMNCs obtained and cultured in vitro in the same culture conditions could be observed after the rhG-CSF injection at 6 hours, but cloning efficiency was (0.50 +/- 0.11) x 10(-6) MNCs and showed statistical difference as compared with control. It is concluded that rhG-CSF to mobilize hemopoietic stem cells can be used to induce mouse MSCs in vivo expansion, which showed the peak value within 6 hours after final injection of rhG-CSF. rhG-CSF have the mini-mobilization effect on murine MSCs derived from bone marrow.