In order to construct generally efficient cell immortalization vector, pTP-hTERT, we modified the traditional piggyBac (PB) transposon using artificial synthesis, PCR and enzyme digestion. The modified vector contained the necessary transposon elements, a PB transposase expression cassette, a co-expression selectable element and a human telomerase reverse transcriptase (hTERT) expression cassette. The co-expression selectable element had two markers, enhanced green fluorescent protein (EGFP) gene and puromycin-resistance (Puro) gene, linked by porcine teschovirus-1 2A peptide (P2A). To validate the functionality of vector elements, we transfected pTP-hTERT into HEK293 cell, selected the positive cell clones and then conducted RT-PCR, Western blotting (WB) and Tail-PCR, methylene blue staining and statistic analysis on selected cells. The results of sequencing and cell culture show that the pTP-hTERT was constructed successfully and the positive cell could be selected by puromycin. The WB results, P2A cutting EGFP and Puro fusion protein with high efficiency, reflected the selectable element worked. The sequencing result of Tail-PCR confirmed the vector integrated into the genome through transposition. The results of methylene blue staining and statistic analysis indicated the clone of positive cells triggered by pTP-hTERT significantly increased (P < 0.01) compared with control group. The construction of pTP-hTERT provides an efficient tool for establishing immortalized cell lines and a demonstration for building other eukaryotic plasmids.
Cell Line ; DNA Transposable Elements ; genetics ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; HEK293 Cells ; Humans ; Plasmids ; Polymerase Chain Reaction ; Telomerase ; genetics ; Transfection

Objective To evaluate the efficacy of combination therapy with finasteride and doxazosin in benign prostatic hyperplasia/ lower urinary tract symptoms (BPH/LUTS) patients with intravesical prostatic profusion (IPP).Methods A total of 322 BPH/LUTS patients who accepted combination therapy with finasteride and doxazosin were enrolled in this study.Patients were divided into 4 groups according to the degree of IPP:group Ⅰ(IPP＞10 mm),group 2 (IPP between 5 mm and 10 mm),group 3 (IPP＜5 mm),control group (without IPP).All patients were received inasteride 5 mg once per day and doxazosin 4 mg once per day for 6 months.International prostate symptom score (IPSS),prostatic specific antigen(PSA),ultrasonographic and urcdynamic evaluation were compared before and after treatment.The correlations between the above factors and IPP were estimated by Logistic regression analysis.Results After 6 month of treament,the changes of IPP degree and the maximal urinary flow rate (Qmax) had no significant differences in group 1,group 2 and group 3 as compared with before treatment (all P＞0.05).The IPSS in group 1 was not significantly different before and after treatment (P＞0.05).There were significant differences in the PSA level,IPSS,total prostate volume (TPV),transition zone volume (TZV),residual urine volume (PVR) in the 4 groups before and after treatment (all P＜0.05).Logistic regression analysis showed that PVR and Qmax had positive and negative correlations with IPP (P＜0.001 and P=0.024),respectively.Conclusions Combination therapy with finasteride and doxazosin can significantly improve the symptoms of LUST and reduce the total prostate volume in patients with BPH/LUTS,but for BPH patients with IPP,the combination therapy can not effectively alleviate the degree of IPP.The increase of residual urine volume and decrease of Qmax may enhance the risk of bladder outlet obstruction in BPH patients with IPP.

Objective To evaluate clinical features, therapeutic regimen and prognosis of unexplained rhabdomyolysis. Method Clinical manifestations, therapeutic regimen and prognosis were recorded in 23 patients,who were admitted to The First Affiliated Hospital of Nanjing Medical University 13 to 27 August,2010.The 23 patients were diagnozed as unexplained rhabdomyolysis. Results The patients all presented myalgia of upper body,like neck,waist and back,maybe with asthenia, nausea,dyspnea,abdominal pain, red urine or changed color of urine. Laboratory examination: obviously step-up of creatine kinase [CK: (4655 ± 2556) U/L( normal: 25 ～ 190U/L) AST:(141 ±78) U/L(normal:10～45 U/L),LDH:(348± 127) U/L(normal: 110～ 250 U/L)]and myoglobin[( Mb ＞ 1000 μg/L (normal: 0 ～ 50 μg/L)]. Therapeutic regimen included treatment of the underlying diseases, volume repletion, alkalization and dealing with the complications. No patients developed acute renal failure.All the patients recovered. Conclusions Rhabdomyolysis is a syndrome with different clinical manifestations.However, early diagnosis, proper treatment could prevent serious complications,and prognosis is good.

OBJECTIVETo seek an effective drug for treatment of human hyperplastic scar through studying the effects of curcumin on fibroblast growth and collagen synthesis.

METHODSFibroblasts derived from scar tissue and from normal epidermal tissue were isolated and cultured separately with tissue-block method, their morphology were observed under invert phase contrast microscope, their growth curve was drawn respectively to determine the speed of growth. Then, fibroblasts from scar were stimulated with curcumin in different concentrations (0, 12.5, 25, 50 and 100 micromol/L) for detecting the inhibitory effect of curcumin on growth of fibroblasts using MTT methods and that on activity of procollagen alpha-1 gene transcription in fibroblast was detected by RT-PCR.

RESULTSThe cell growth curve showed that double-multiplying time was 5 days in fibroblasts from scar and 4 days in those from normal dermis, showing significant difference between them (P < 0.05). MTT showed that curcumin in 12.5 micromol/L showed a cell proliferation enhancing trend, and its absorbance value was significantly higher than that in the normal group, but the effect turned to inhibition when concentration increased to over 25-100 micromol/L, and became significant inhibition at concentration of 50 and 100 micromol/L. Besides, curcumin also showed markedly inhibition on collagen type I synthesis in fibroblasts (P < 0.01).

CONCLUSIONHigh concentration curcumin can inhibit effectively the fibroblast proliferation and collagen I synthesis in hyperplastic scar, therefore, may has therapeutic effect on the disease in human being.

Cell Proliferation ; drug effects ; Cells, Cultured ; Cicatrix, Hypertrophic ; metabolism ; pathology ; Collagen Type I ; biosynthesis ; Curcumin ; pharmacology ; Fibroblasts ; drug effects ; metabolism ; pathology ; Humans

Objective To determine the influence of intense pulsed light (IPL) on the secretion of TGF-β1 in cultured human fibroblasts and the intervention of JNK inhibitor.Methods The callan foreskin fibroblasts were cultured and divided into 2 groups. In the IPL treatment group, cells were irradiated with IPL with fluences of0 (negative control), 10, 18, 27, 36, and 36 J/cm2 × 2 (irradiated with IPL with fluences of 36 J/cm2 twice). In the IPL + inhibitor group, cells were irradiated with IPL with fluences of 36 J/cm2 after incubation with the inhibitor SP600125 for 2 h. TGF-β1 in the culture supernatant was evaluated 48 h after the irradiation using enzyme-linked immunosorbent assay. Results Compared with the negative control, TGF-β1 in the culture supernatant decreased at the IPL irradiation of 10, 18, 27, and 36 J / cm2, whereas TGF-β1 increased at the IPL irradiation of 36 J/cm2× 2. In the IPL + inhibitor group, the concentration of TGF-β1 in the culture supernatant decreased compared with the controls (P<0.05). Conclusion IPL can suppress the secretion of TGF-β1 at the lower fluence and promote the secretion at a higher fluence. JNK inhibitor may play an inhibitive role when IPL regulates the TGF-β1 secretion in cultured human fibroblasts. IPL may stimulate TGF-β1 secretion of the fibroblast cells in human skin via JNK signal pathway.

In order to search for new antiplatelet agents with higher potency, a series of tetramethylpyrazine ( TMP) /chalcone hybrids ( 2-26) were synthesized and evaluated based on the principle of bioisostere and hybrid-ization. They exerted inhibitory activity against adenosine diphosphate ( ADP )-induced and arachidonic acid ( AA)-induced platelet aggregation to varied extent. Among them, compound 8 was the most potent with IC50 of 0. 14 mmol/L on ADP-induced platelet aggregation ( 9. 1 folds of TMP and 10. 5 folds of chalcone ) and 0. 09 mmol/L on AA-induced platelet aggregation ( 8. 8 folds of TMP and 10. 0 folds of chalcone) , which was superior to clinically used anti-platelet drug aspirin ( ASP, IC50 =0. 15 mmol/L) .

A site-directed mutant DNA fragment was synthesized and transfected into clinical Neisseria gonorrhoeae (NG) stains to construct the transformants that contained the corresponding mutagenesis of regulation region of mtrR gene. According to the technique of gene splicing by overlap extension (SOEing), a DNA segment with specific mutagenesis was constructed by two-step polymerase chain reaction (PCR). The mutation fragments EF could be used for the next experiment in which the mutation NG strains were induced. By comparing the recombinant EF fragments to the corresponding DNA fragments of clinical NG strains, 2 of these were not compatible completely. The results of sequencing revealed that there was a 9 bp deletion between the 45 to 54 inverted repeat sequence localized within the mtrR promoter. It can be confirmed that the fragments EF are the specifically designed mutant fragments.
Bacterial Proteins/*genetics ; DNA Fragmentation ; DNA, Bacterial/genetics ; Mutagenesis, Site-Directed ; Neisseria gonorrhoeae/*genetics ; Neisseria gonorrhoeae/metabolism ; Recombination, Genetic ; Repressor Proteins/*genetics ; Sequence Deletion ; Transfection ; Transformation, Bacterial

Objective To investigate the effect of catch-up growth on insulin resistance(IR) through analysis of biochemical and metabolic indices in premature infants.Methods There were 126 infants admitted in the Department of Neonatology,the Affiliated Hospital of Guiyang Medical College from December 2010 to December 2013 [factors which might affect the secretion of insulin(INS),C peptide and insulin-like growth factor-1 (IGF-1) were excluded].According to gestational age and birth weight,babies were divided into small for gestational age (SGA) group and appropriate for gestational age (AGA) group.And according to the age on follow-up,babies were divided into 1-to-6-month-old group,7-to-12-month-old group and ≥ 1-to-3 year-old group.All cases had 6 mL peripheral venous blood sampled in the early morning during fasting in the first week,and 6,12,24,36 months after birth.They were tested for levels of INS,C peptide,IGF-1,triglyceride (TC),total cholesterol (TG),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C),fasting blood-glucose (FBG),albumin,prealbumin and creatinine respectively.At the same time,the physical parameters were measured,including weight,body length,and calculated insulin resistance index (IRI),Ponderal index,weight standard deviation score (SDS),and length SDS.Results (1) Catch-up growth after premature birth occupied 65.6％ (63/96 cases),whereas no catch-up growth occupied 34.4％ (33/96 cases) of study snbjects,and among them catch-up growth of 37 cases was better (8 cases of SGA,29 cases of AGA),26 cases showing catch-up growth(7 cases of SGA,19 cases of AGA),33 cases without catch-up growth(11 cases of SGA,22 cases of AGA).No statistical significance was found in the distribution of catch-up growth between SGA group and AGA group(P ＞ 0.05).(2) The LgIRI,LgINS of group with good catch-up growth was significantly lower than the group with no catch-up growth group (F =3.55,3.47) in infancy,but the level of IGF-1 and prealbumin was higher than that of no catch-up growth group (F =3.55,4.94),the difference had statistical significance (P ＜ 0.05) ; the better catch-up growth was associated with higher IGF-1 and prealbumin,but with lower LgIRI.(3) The risk factors for insulin resistance were SGA (OR =7.904,P =0.001),low birth weight ＜ 1 500 g (OR=8.737,P=0.019),and no catch-up growth (OR=11.706,P=0.000).Conclusions The better catch-up growth in infancy is associated with higher IGF-1 and prealbumin,but lower IR.The risk factors of IR include SGA,low birth weight and no catch-up growth,and the last being the major factor.

Objective To study the dosimetry for copper compensator in IMRT.Methods 10 patients (3 nasopharyngeal carcinoma,4 esophageal cancer,3 colorectal cancer) from IMRT plans made in Oncentra Planning System enrolled in this study.The fluence matrices of each plan were converted into compensator thickness matrices based on attenuation coefficient of copper under 6 MV radiation ray which was measured with ion chamber.While irradiating the homogenous phantom with the intensity modulated plans performed by copper compensator,film was used to measure the planar dose.In the end,γanalysis was carried out between the measured and calculated planar doses with the criteria of 3％ DD/3 mm DTA.Paired t test to MU differences of MLC plans and copper compensator plans.Results The γanalysis results showed the planar dose pass rates of 10 plans lie between 90.2％ and 98.2％,which means the IMRf technique based on copper compensator can meet the clinical needs.The MU for copper compensator IMRT plans is less than the MLC IMRT plans (873.9 vs.975.1,P =0.005).Conclusions Compared to conventional IMRT based on MLC,copper compensator has the advantage of lower requirement of hardware and lower scattering dose in patient.Copper compensator can meet the needs of clinical practice.