Objective To construct the recombinant adeno-associated virus vector(rAAV) expressing the human tissue kallikrein(HK)gene and to detect the expression of interested gene in human umbilical vein endothelial cells(HUVEC)which were infected with different titers of rAAV. Methods The HK gene was cloned into the pAAV-MCS and co-transfected AAV-293 cells with other two plasmids(the pAAV-RC,and pHelper)by lipofectamine.The recombinant AAV(rAAV HK)particles was harvested and its viral titer was measured.HUVEC were infected with different titers of rAAV-HK particles.Seventy-two hours later,the expression of the interested gene was detected by RT-PCR and ELISA.Results The AAV expression system of human tissue kallikrein gene was successfully constructed.The viral titer of recombinant AAV reached 6.2?10~7 particles/ml. When compared with the control group,the transcription of HK gene in the group which was infected with rAAV-HK increased significantly[(0.59?0.12)vs(0.26?0.05)(P＜0.05)],and the expression of HK in the HUVEC was three times more than that in the control[(120.1?40.9)vs (30.8?12.8)](P＜0.001).The transcription of eNOSmRNA in the infected HUVEC was higher than that of the control[(1.19?0.28)vs(0.66?0.11)](P＜0.05).The transcription of caspase-3 mRNA was lower than that of the control[(0.30?0.25)vs(0.67?0.27)](P＜0.05).And there was no obvious variation happened in the transcription of VEGF,ET-1,TR-B,bradykinin B1 receptor and Bradykinin B2 receptor.Conclusions When co-transfected AAV-293 cells with three plasmids (pAAV-HK,pAAV-RC,pHelper),the HK gene expression is significantly and stably increased. Introducing HK gene into HUVEC can improve endothelial transfection efficiency.

Objective Tissue kallikrein cleaves kininogen substrate to produce vasoactive kinin peptides that have been implicated in the proliferation of vascular smooth muscle cells. We investigated the effects of adenovirus-mediated human tissue kallikerin(Ad-hKLK1)gene delivery on the proliferation of vascular smooth muscle cells of SHR(VSMCsSHR)induced by platelet derived growth factor-BB(PDGF-BB). Methods Primary VSMCsSHR were isolated and cultured from thoracic aorta of male SHR. The VSMCsSHR proliferation induced by PDGF-BB was accessed by cell counting and methyl thiazolyl tetrazoliuin(MTT). Western blot was used to determine the protein expression of hKLK1, the cycle-independent kinase inhibitors p27Kip1 and p21Cip1 . The mRNA expressions of bradykinin B1 receptor and B2 receptor were detected by RT-PCR in VSMCsSHR. Results Proliferation of VSMCsSHR induced by PDGF-BB was significantly inhibited post transfection of Ad-hKLK1(20 - 100 MOI)in a MOI-dependent manner. The peak inhibition titer of Ad-hKLK1 was 100 MOI with peak inhibition rate of 39.3%(cell counting, n = 3,P ＜0.01), 30.2%(MTT, n-3 ,P ＜ 0.01)and 36.4%(peak stunning rate of cell-cycle in phase G0/G1). The inhibitory effects of proliferation and cell-cycle caused by hKLK1 gene delivery could be abolished by Hoe140, a bradykinin B2 receptor antagonist. The protein expression of p27Kip1 and p21Cip1 increased significantly after the hKLK1 gene delivery, whereas Hoe140 nearly completely blocked these effects(n = 3,P ＜0.001 ,respectively). PDGF-BB also significantly upregulated the mRNA expression of B2 receptor but not B1 receptor in VSMCsSHR. Conclusion The hKLK1 gene delivery could inhibit PDGF-BB induced proliferation in VSMCsSHR through Bradykinin B2 receptor and up-regulate expression of p27Kip1 and p21Cip1.

Objective To analyze the resignation status quo of contracted nurses, so as to establish an effective management mechanism and system for them. Methods 123 contracted nurses who resigned from our hospital from 2005 to 2007 were selected, collecting their individual skill files and undertaking the statistics analysis for the reasons. Results The rate of resignation was from 10.2% to 11.2%. Among the resigned contracted nurses, 62.5% of them resigned before 25 years old;The rate of initiative resignation decreased gradually the three late years (P＜0.05);86.2% of the contracted nurses resigned in the first 5 years and 48% of them were nurses of 1～2 years;The flint three reasons of resignation were changing employment (40%), individual reasons to hometown (29%), and incompetence for occupation (13%);It was an inverse ratio between the working lifetime and the number of resigned contracted nurses who broke the promises.Conclusions It is necessary to formulate the employment specification that suits the hospital feature under the new laber contract law so as to deplete or to avoid the initiative resignation of the contracted nurses.

OBJECTIVETo study whether hematologic malignancy patients with anemia have a lower erythropoietin (EPO) response.

METHODSSerum EPO levels were detected by ELISA in patients with hematologic malignancies and with iron deficiency anemia (IDA). Eighty patients with hematologic malignancies, including 13 multiple myeloma (MM), 7 chronic lymphocytic leukemia (CLL) and 60 non-Hodgkin's lymphoma (NHL) were studied. Thirty of them had anemia(21 NHL,6 MM and 3 CLL). Twenty patients with IDA were studied as the control.

RESULTSHematologic malignancy patients with anemia had higher EPO levels [(97.8 +/- 183.9) IU/L] than those with normal Hb values [(27.8 +/- 85.4) IU/L; P <0.01]. In patients with IDA, serum EPO response was inversely correlated with Hb level (r= -0.5, P <0.05) , but no such inverse correlation was found in the hematologic malignancy patients with anemia (r = -0.14). After corrected for Hb level, the serum EPO levels were significantly lower in anemic patients with hematologic malignancies than in IDA patients (P = 0.032) , indicating a decreased EPO response in the former group.

CONCLUSIONAnemia associated with hematologic malignancy might result from an inappropriately low EPO response. EPO treatment for these patients may be beneficial.

Adolescent ; Adult ; Aged ; Anemia, Iron-Deficiency ; blood ; complications ; Enzyme-Linked Immunosorbent Assay ; Erythropoietin ; blood ; Female ; Hematologic Neoplasms ; blood ; complications ; Hemoglobins ; metabolism ; Humans ; Male ; Middle Aged ; Prospective Studies

Objective:To investigate the changes of CTCs and the correlation between the changes of CTCs and the clinical efficacy of neoadjuvant immunotherapy for non-small cell lung cancer (NSCLC).Methods:A retrospective case-control study was conducted to collect the data of 23 patients with NSCLC who received neoadjuvant immunotherapy in the Third Xiangya Hospital from June 2018 to December 2019. They were 35-76 years old with a median age of 52 years old, including 13 male patients and 10 female patients. The CTCs value, evaluation results from response evaluation criteria in solid tumor (RECIST) and major pathological response were evaluated before treatment, after neoadjuvant immunotherapy and after operation. Mann Whitney U test was used for the comparison between the two groups, Wilcoxon test was used for the comparison of association samples, and Kruskal Wallis test was used for the comparison between multiple samples.Results:The CTCs value was positively correlated with tumor progression, that the CTCs value of ⅡB group, ⅢA group and ⅢB group was 10.69 (3.87) FU/3 ml, 12.90 (2.24) FU/3 ml and 16.04 (3.43) FU/3 ml, and the difference was statistically significant (χ 2=7.829, P=0.020). Then CTCs decreased to 7.60(4.79) FU/3 ml significantly ( Z=4.197, P=0.000), and decreased to 6.22(2.80) FU/3 ml significantly again after surgery( Z=-2.950, P=0.005). In RECIST results, the CTCs value of CR group, PR group and SD group was 12.90(3.79)FU/3 ml, 12.52(3.96) FU/3 ml and 13.58(5.11) FU/3 ml,and no significant difference before treatment (χ2=1.806, P=0.405). After neoadjuvant immunotherapy, the CTCs of CR group decreased to 6.22(3.87) FU/3 ml significantly ( Z=-4.950, P= 0.000), and also PR group to7.32(4.31) FU/3 ml ( Z=-3.180, P=0.001) or SD group to ( Z=-2.023, P=0.043). There was no significant difference between CR group and PR group ( Z=-0.838, P=0.402), but significant difference between SD group and CR/PR group ( Z=-1.922, P=0.050). After operation, the CTCs of CR, PR and SD group decreased to 6.09(3.43) FU/3 ml, 6.40(1.82) FU/3 ml and 9.20(5.16) FU/3 ml,and there was no significant difference to preparation in CR group and PR group, but significant difference in SD group ( Z=-2.023, P=0.043). There was no significant difference between CR group and PR group ( Z=-1.134, P=0.257), but significant difference between SD group and CR/PR group ( Z=-1.624, P=0.014). Before treatment,CTCs of MPR group and non-MPR group were 11.98(4.14) FU/3 ml and 13.54(4.76) FU/3 ml,and there was no significant difference between them ( Z=-1.354, P=0.176). After neoadjuvant immunotherapy, the CTCs of MPR group decreased to 6.36(2.65) FU/3 ml significantly ( Z=-2.934, P=0.001) and also in non-MPR group to 10.88(2.80) FU/3 ml ( Z=-2.840, P=0.003); but there was significant difference between MPR group and non-MPR group ( Z=-3.693, P=0.000), and also the change of CTCs between two groups ( Z=-2.770, P=0.006). After operation, the CTCs of MPR group decreased to 5.40(1.33) FU/3 ml insignificantly ( Z=-0.533, P=0.594) but significantly to 7.05(3.80) FU/3 ml in non-MPR group ( Z=-2.734, P=0.030), and significant difference between them ( Z=-1.900, P=0.011). Conclusion:The value of CTCs is negatively correlated with the efficacy (RECIST and MPR) of neoadjuvant immunotherapy for NSCLC, which can be used for clinical efficacy evaluation of neoadjuvant immunotherapy.

Objective:To investigate the changes of CTCs and the correlation between the changes of CTCs and the clinical efficacy of neoadjuvant immunotherapy for non-small cell lung cancer (NSCLC).Methods:A retrospective case-control study was conducted to collect the data of 23 patients with NSCLC who received neoadjuvant immunotherapy in the Third Xiangya Hospital from June 2018 to December 2019. They were 35-76 years old with a median age of 52 years old, including 13 male patients and 10 female patients. The CTCs value, evaluation results from response evaluation criteria in solid tumor (RECIST) and major pathological response were evaluated before treatment, after neoadjuvant immunotherapy and after operation. Mann Whitney U test was used for the comparison between the two groups, Wilcoxon test was used for the comparison of association samples, and Kruskal Wallis test was used for the comparison between multiple samples.Results:The CTCs value was positively correlated with tumor progression, that the CTCs value of ⅡB group, ⅢA group and ⅢB group was 10.69 (3.87) FU/3 ml, 12.90 (2.24) FU/3 ml and 16.04 (3.43) FU/3 ml, and the difference was statistically significant (χ 2=7.829, P=0.020). Then CTCs decreased to 7.60(4.79) FU/3 ml significantly ( Z=4.197, P=0.000), and decreased to 6.22(2.80) FU/3 ml significantly again after surgery( Z=-2.950, P=0.005). In RECIST results, the CTCs value of CR group, PR group and SD group was 12.90(3.79)FU/3 ml, 12.52(3.96) FU/3 ml and 13.58(5.11) FU/3 ml,and no significant difference before treatment (χ2=1.806, P=0.405). After neoadjuvant immunotherapy, the CTCs of CR group decreased to 6.22(3.87) FU/3 ml significantly ( Z=-4.950, P= 0.000), and also PR group to7.32(4.31) FU/3 ml ( Z=-3.180, P=0.001) or SD group to ( Z=-2.023, P=0.043). There was no significant difference between CR group and PR group ( Z=-0.838, P=0.402), but significant difference between SD group and CR/PR group ( Z=-1.922, P=0.050). After operation, the CTCs of CR, PR and SD group decreased to 6.09(3.43) FU/3 ml, 6.40(1.82) FU/3 ml and 9.20(5.16) FU/3 ml,and there was no significant difference to preparation in CR group and PR group, but significant difference in SD group ( Z=-2.023, P=0.043). There was no significant difference between CR group and PR group ( Z=-1.134, P=0.257), but significant difference between SD group and CR/PR group ( Z=-1.624, P=0.014). Before treatment,CTCs of MPR group and non-MPR group were 11.98(4.14) FU/3 ml and 13.54(4.76) FU/3 ml,and there was no significant difference between them ( Z=-1.354, P=0.176). After neoadjuvant immunotherapy, the CTCs of MPR group decreased to 6.36(2.65) FU/3 ml significantly ( Z=-2.934, P=0.001) and also in non-MPR group to 10.88(2.80) FU/3 ml ( Z=-2.840, P=0.003); but there was significant difference between MPR group and non-MPR group ( Z=-3.693, P=0.000), and also the change of CTCs between two groups ( Z=-2.770, P=0.006). After operation, the CTCs of MPR group decreased to 5.40(1.33) FU/3 ml insignificantly ( Z=-0.533, P=0.594) but significantly to 7.05(3.80) FU/3 ml in non-MPR group ( Z=-2.734, P=0.030), and significant difference between them ( Z=-1.900, P=0.011). Conclusion:The value of CTCs is negatively correlated with the efficacy (RECIST and MPR) of neoadjuvant immunotherapy for NSCLC, which can be used for clinical efficacy evaluation of neoadjuvant immunotherapy.

Objective:To construct HIV-1 pseudovirus containing enhanced green fluorescent protein ( EGFP ) gene. To understand the interaction between the virus and the cells. Methods: HIV-1 pseudovirus containing EGFP gene was constructed by lentiviral packaging systems, and its EGFP gene was amplified using RT-PCR. The level of genomic integration and transcription of HIV-1 pseudovirus containing EGFP gene were detected on iDCs infected with HIV-1 pseudovirus. At the same time, research on expression of the EGFP gene in iDCs infected with HIV-1 pseudovirus was performed. Results:The EGFP gene of HIV-1 pseudovirus was detected through RT-PCR. The EGFP gene was identified in iDCs infected with HIV-1 pseudovirus through PCR and RT-PCR. The EGFP was observed in iDCs infected with HIV-1 pseudovirus under fluorescence microscopy. Conclusion: HIV-1 pseudovirus containing EGFP gene has been successfully produced. The HIV-1 pseudovirus that we constructed can infect iDCs,then its RNA can integrate into the genome of iDCs in the way of reverse transcription,and the EGFP gene could express in the iDCs after infected with HIV-1 pseudovirus.

BACKGROUND: Acute liver failure (ALF) caused by viral and non-viral hepatitis is often accompanied with severe metabolic disorders, the accumulation of toxic substances and continuous release and accumulation of a large number of endogenous toxins and inflammatory mediators. The present study aimed to investigate the effects of various combined non-biological artificial liver treatments for patients with acute liver failure (ALF) complicated by multiple organ dysfunction syndrome (MODS). METHODS: Thirty-one patients with mid- or late-stage liver failure complicated by MODS (score 4) were randomly divided into three treatment groups: plasmapheresis (PE) combined with hemoperfusion (HP) and continuous venovenous hemodiafiltration (CVVHDF), PE+CVVHDF, and HP+CVVHDF, respectively. Heart rate (HR) before and after treatment, mean arterial pressure (MAP), respiratory index (PaO2/FiO2), hepatic function, platelet count, and blood coagulation were determined. RESULTS: Signifi cant improvement was observed in HR, MAP, PaO2/FiO2, total bilirubin (TBIL) and alanine aminotransferase (ALT) levels after treatment (P<0.05). TBIL and ALT decreased more signifi cantly after treatment in the PE+CVVHDF and PE+HP+CVVHDF groups (P<0.01). Prothrombin time (PT) and albumin were signifi cantly improved only in the PE+CVVHDF and PE+HP+CVVHDF groups (P<0.05). TBIL decreased more significantly in the PE+HP+CVVHDF group than in the HP+CVVHDF and PE+CVVHDF groups (P<0.05). The survival rate of the patients was 58.1％ (18/31), viral survival rate 36.4％ (4/11), and non-viral survival rate 70％ (14/20). CONCLUSION: Liver function was relatively improved after treatment, but PE+HP+CVVHDF was more efficient for the removal of toxic metabolites, especially bilirubin. The survival rate was significantly higher in the patients with non-viral liver failure than in those with viral liver failure.

Objective To investigate the effects of human tissue kallikrein 1 (Ad-hKLK1) gene delivery on the neointima formation in carotid arteries of spontaneously hypertensive rats (SHRs). Methods Carotid artery restenosis was induced in male SHR rats by balloon-injury. Rats were randomly assigned into 4 groups: Sham-operated (n = 6); Angioplasty (phosphate buffered solution 50 μl, n = 8); Vector virus (control virus, 1×10~9 IU in 50 μl, n =8) and Ad-hKLK1 (Ad-hKLK1, 1×10~9 IU in 50 μl, n =8). Rats were sacrificed 4 weeks later. The wall-to-lumen area ratio and intima/media ratio in carotid artery were assessed by image analysis in HE stained sections. The mRNA bradykinin receptor(B1R and B2R) expressions were detected by RT-PCR. The protein expression of the cycle-independent kinase inhibitors p27Kip1 and p21Cip1 were determined by Western blot analysis. Results Wall-to-lumen area ratio reduced 35.6% and intima/media ratio reduced 38.8% in Ad-hKLK1 treated SHRs compared to angioplasty group (all P <0.001). The expression of p27Kip1 and p21Cip1 increased significantly in Ad-hKLK1 treated SHRs compared with angioplasty rats (all P <0.001). The mRNA expression of B2R was significantly upregulated in angioplasty rats compared with sham-operated rats (P <0.05) while mRNA expression of B1R was similar between the 2 groups. Conclusion hKLK1 gene delivery may effectively reduce neointimal formation via downregulating bradykinin B2R and up-regulating the expressions of p27Kip1, p21Cip1 signaling pathways in carotid arteries of SHRs after balloon injury.

Adolescent ; Adult ; Aged ; Anemia ; drug therapy ; Enzyme-Linked Immunosorbent Assay ; Erythropoietin ; blood ; therapeutic use ; Female ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell ; complications ; Lymphoma, Non-Hodgkin ; complications ; Male ; Middle Aged ; Multiple Myeloma ; complications