A mammalian expression plasmid pcDNA3. 1-hCC10 was constructed and identified, then CC10 protein expression in A549 lung cancer cell line was detected. A 273 bp cDNA fragment was amplified from the total RNA of normal lung tissue by using RT-PCR and cloned into expression plasmid cDNA3. 1, and the recombinant plasmid was identified by employing double digestion restriction enzymes Hind III and BamH I and the cDNA sequence was assayed by the Sanger dideoxy-mediated chain termination method. The segment was then transfected into the A549 lung cancer cell line. The protein expression of CC10 was detected by immunofluorescence and Western blot. Our results showed that the cDNA fragment included the entire coding region (273 bp). The recombinant eukaryotic cell expression vector of pcDNA3. 1-hCC10 was successfully constructed, and the sequence of the insert was identical to the published sequence. A549 cells line transfected with the pcDNA3. 1-hCC10 expressed high level of CC10 protein. The recombinant plasmid cDNA3. 1-hCC10 may serve as an effective tool for the study of tumorogenesis and tumor treatment.
Adenocarcinoma/*metabolism ; Adenocarcinoma/pathology ; Cell Line, Tumor ; Genetic Vectors ; Lung Neoplasms/*metabolism ; Lung Neoplasms/pathology ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; Transfection ; Uteroglobin/biosynthesis ; Uteroglobin/*genetics

Objective To set up the reference standard of cyclovirobuxine D.Methods Thermal analysis,HPLC/MS,HPLC with terminal wavelength,HPLC with fluorescence derivation and with ultraviolet derivation,TLC and nonaqueous titration methods were applied to determine the content of cyclovirobuxine D control.Results Thermal analysis can not be used to analyse the purity of cyclovirobuxine D ,and HPLC/MS,HPLC with terminal wavelength,HPLC with fluorescence derivation and HPLC with ultraviolet derivation can obtain the same purity.Conclusion The methods used for the assay of cyclovirobuxine D control were practical.

Cell-free protein synthesis (CFPS) systems based on crude cell extracts have been used in protein expression in vitro. With the researchers' endeavor for decades, the CFPS system has been developed as an important research tool in many frontiers of fundamental and applied biology because of its clear genetic background and simplicity to control the reaction. The yield of CFPS systems derived from prokaryote or eukaryote has increased to several grams per liter with constantly decreasing cost. Nowadays grams of protein could be prepared using a large-scale cell-free system. Recently, the advantages on the expression of complicated, toxic and membrane proteins have shown the great potential of the CFPS systems. The rapid progress of this technology made us to believe that it will take an important place in biopharmaceutical industries undoubtedly.
Bioengineering ; trends ; Cell-Free System ; Drug Industry ; trends ; Protein Biosynthesis

Objective To evaluate the applicable value of two - dimensional speckle tracking imaging on assessing the local function and coronary collateral circulation of acute myocardial ischemia in dogs.Methods Open-chest model to induce acute myocardial ischemia in 10 dogs was established by ligating their anterior descending coronary artery(LAD). Two-dimensional dynamic gray- scale images of two standard left ventricular short axis views at the levels of mitral annulus and papillary muscle were acquired for the off -line comparative analysis before the operation and 0, 30, 60, 120, 240 and 360 minutes after the operation respectively. Peak systolic circumferential strain(CS), peak systolic radial strain(RS) and left ventricular fraction shortenting(LVFS) of 12 segments at the levels of mitral annulus and papillary muscle were analyzed with QLAB software. After the lab experiment,the fresh hearts of dogs were dyed by TTC.Results ①Compared with the preoperative value , the peak systolic CS and RS of ischemia myocardial regions(anteroseptum,anterior and lateral) in left ventricular short-axis decreased significantly( P ＜0. 05)during 30 to 60 minutes after ligating LAD. Sixty minutes later, the peak systolic CS and RS had a tendency to return to the level before the operation,although the statistical difference was existing. The peak systolic RS and CS also decreased in certain nonischemic regions (inferolateral and inferior). But CS and RS in inferoseptal regions didn't alter significantly. ②Compared with the preoperative value,LVFS in the ischemia regions were significantly decreased( P ＜0.05), while nonischemic regions had no significant difference. ③The results of TTC dyeing showed that the infarcted regions in left ventricular short-axis were anteroseptum, anterior and lateral with blood supplied by LAD, which were consistent with the results of RS and CS. Conclusions RS and CS,as the parameters of myocardial strain may reflect the range and extent of acute myocardial ischemia,and the strain changes of local myocardial segments after the construction of coronary collateral circulatory.

Objective To explore the expression of ribosomal protein L6 (RPL6) in prostate cancer and its clinical sig-nificance. Methods RT-qPCR and Western blot assay were used to measure the mRNA transcription and protein expres-sion levels of RPL6 in prostate cancer tissues (n=80) and adjacent non-cancerous tissues (n=62). The relationship between RPL6 mRNA expression level and clinicopathological factors of prostate cancer was statistically analyzed. Results The mRNA and protein expression levels of RPL6 were significantly higher in prostate cancer tissues compared with those of non-cancerous tissues (P<0.05). There were higher serum expression levels of prostate specific antigen (PSA) and higher Gleason score in prostate cancer tissues. The expression level of RPL6 mRNA was significantly higher in patients with lymph node metastasis and late clinical stage (P<0.05). There were no significant differences in PSA levels between different ages, with or without seminal vesicle invasion and different surgical margin status (P>0.05). Kaplan-Meier survival analysis of biochemical recurrence (BCR)-free survival time showed the significantly lower recurrent rate in patients with high RPL 6 mRNA expression(χ2=4.530,P=0.033). Conclusion The elevated expression of RPL6 may play a role in the development of prostate cancer, and which can be used as a tumor marker to assess the prognosis of prostate cancer.

Objective To establish the method of contact heat evoked potential(CHEP)and to explore the value of this evoked potential in pain testing of patients with cerebral infarction.Methods A total of 100 healthy volunteers and 30 patients were examined.The healthy volunteers were divided into 3 groups according to the length of their arms:(Group A:56.0～65.0 cm ;Group B :65.5～74.0 cm ;Group C :74.5～83.0 cm).A recently de- veloped heat-foil technique with a rapid temperature rising rate at 70℃/s was used to elicit pain and contact heat e- voked potentials.Contact heat was delivered via one circular thermode(diameter 27 mm,area 573 mm~2)and set at two intensity levels(49.5℃and 54.5℃)to three body sites:the thenar eminence,the dorsum of hand and proximal volar forearm.The subjects were asked to rate the pain with numerical rating scale after each stimulus and CHEP was recorded from Cz and Pz.The association between stimulus intensities and pain rating was explored,the main compo- nents of the evuked potential were watched.CHEP,sensory conduction velocity(SCV)and somatosensory evoked potentials(SEP)were performed in patients with hemi-anesthesia caused by cerebral infarction.Results The pain intensity ratings were 3.2?0.3 and 4.4?0.5 at thenar eminence,5.0?0.7 and 6.3?0.8 at the dorsum of hand and 5.3?0.6 and 7.2?0.5 at the proximal volar forearm when the temperature of 49.5℃and 54.5℃was applied, respectively;Three components,Cz/N550,Cz/P750 and Pz/P1000,were identified in the evoked potentials.Cz/ N550 and Cz/P750 appeared when the dorsum of hand and proximal volar forearm were stimulated.In contrast,Pz/ P1000 could be identified when nociceptors of thenar eminence and proximal volar fbrearm were excited.In the pa- tients with cerebral infarction,CHEP disappeared or became abnormal on one side,while SCV and SEP were normal on that side.Conclusion It was suggested that CHEP could be elicited reliably in the controls.CHEP is helpful in the assessment of analgesia in patients with cerebral infarction.

Objective To evoke cerebral potentials by stimulating nociceptive fibers with contact heat evoked potentials stimulator (CHEPS)and estimate the nerve conduction velocities of peripheral nerve fibers mediating these responses.Methods Subjects were set in supine position.A heat-foil technology with a rapid rising speed at 70 ℃/s was used to elicit pain and contact heat evoked potentials(CHEP).Contact heat was delivered via one circular thermode (diameter 27 mm,area 573 mm~2).Thermal stimuli were sent at two intensity levels (49.5 ℃ and 54.5 ℃) to three body sites:thenar eminence,the dorsum of hand and proximal volar forarm.Contact heat evoked potentials were recorded from Cz and Pz.A systemic effect between stimulus intensities and pain rating were observed,the main components of this evoked potential were observed.Nerve conduction velocity was calculated from latency difference of CHEP and center to center distance of distal and proximal stimulus arrays.Results The pain intensity rating was 3.2?0.3 and 4.4?0.5 when thenar eminence was stimulated at the temperature of 49.5 ℃ and 54.5 ℃ respectively;the rating was 6.3?0.8 and 7.2?0.5 when the dorsum of hand and proximal volar forarm were stimulated at the temperature of 54.5 ℃ respectively.Three components,Cz/N550,Cz/P750 and Pz/P1000,were found in the evoked potentials.Nerve conduction velocities of the fibers were (12.9?7.5) and (1.7?0.4) m/s respectively,which were corresponding to those of A8 fiber and C fiber.Conclusions CHEPs can be elicited reliably and stably.Velocities of peripheral nerve fibers demonstrate that A8 fiber and C fiber mediate the response.

Bacterial Infections ; epidemiology ; microbiology ; China ; Drug Resistance, Bacterial ; Humans ; Microbial Sensitivity Tests

Objective To investigate the effect and mechanism of STAT3 gene silencing on the growth of xenografts in human pancreatic cancer SW1990 cells in nude mice.Methods The expression vector inserted with shRNA targeting at STAT3 gene was constructed and was stably transfected into SW1990 cells (SW1990-RNAi group).SW1990 cells transfected with negative control shRNA expression vector (SW1990-Con group) and parent SW1990 (SW1990 group) were used as controls.STAT3,VEGF,MMP-2 protein expressions in these groups were determined by using Western blot.The subcutaneous xenografts models were established in nude mice,and the growth of xenografts was observed,CD34 expressions were determined by immunohistochemistry and MVD was measured.Results The expression of STAT3 protein was 84.69 ± 9.31,82.00 ± 7.76,7.93 ± 1.24,repectively,in SW1990 group,SW1990-Con group,SW1990-RNAi group,and the expression of VEGF protein was 82.94 ± 8.97,80.86 ± 10.28,39.04 ± 6.23,respectively,and the expression of MMP-2 protein was 40.88 ± 5.09,38.26 ± 5.71,12.54 ± 2.15,respectively.The expression in SW1990-RNAi group was significantly lower than those in other 2 groups (P ＜ 0.05),while the expression of all three proteins between SW1990-Con group and SW1990 group was not significantly different.The weight of the xenografts in SW1990 group,SW1990-Con group,SW1990-RNAi group was (2.2 ± 0.4),(2.2 ± 0.3),(0.5 ± 0.3) g,respectively ; the MVD of the xenografts was (20.35 ± 2.41),(18.79 ± 1.94),(9.62 ± 1.06) per high power field,respectively,and the number in SW1990-RNAi group was significantly lower than those in other 2 groups (P ＜ 0.05 or P ＜ 0.01),while the difference between SW1990-Con group and SW1990 group were not significant.Conclusions Inhibition of STAT3 gene expression can significantly slow the growth of SW1990 xenografts in nude mice,and the mechanism may be related with down-regulation of VEGF and MMP-2 expression and inhibition of the angiogenesis of pancreatic cancer.

Objective To investigate the effects of AstragalosideⅣ (AST) on the expression of high glucose peritoneal dialysis solution (PDS)-induced transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF) in cultured human peritoneal mesothelial cells. To discuss the regulating effect of AST on induced fibrosis cytokines.Methods The HMrSV5 cell line was cultivated to the fifth generation and divided into normal group (10%FBS cultivation solution), model group (4.25% PDS and 10% FBS cultivation solution) and the low, medium and high doses AST groups (4.25% PDS with a respective 10, 20, 40 μg/mL AST). MTT colorimetric assay was employed to detect cell activity and ELISA was applied to detect expression of TGF-β1, CTGF and VEGF in cultured supernatants.Results Except for 5 μg/mL group, HPMCs activity of high glucose plus different concentration AST groups were enhanced in different degrees, especially with 40, 45, and 50 μg/mL (P<0.05). The expression of TGF-β1, CTGF, and VEGF in model group increased. Compared with the control group, expression of the three AST groups significantly decreased and showed dose-effect relationship (P<0.05). Conclusion AST could reduce the expression of TGF-β1, CTGF and VEGF in high glucose-induced HPMCs and was useful in slowing down the progress of peritoneal fibrosis.