[Calibration on chromone reference extract and application on quality control of Saposhnikoviae Radix].

The calibration of chromone reference extract(CRE) was conducted and a quality control method of Saposhnikoviae Radix(SR) was established based on CRE. Meanwhile, the quality control system of SR was improved and the feasibility of using reference extract as a substitute for single reference substance in quality control of Chinese medicine was discussed. In this study, the content of the prepared CRE was calibrated with prim-O-glucosylcimifugin, cimifugin, 4'-O-ß-D-glucosyl-5-O-methylvisamminol, and secO-glucosylhamaudol as indicators. Subsequently, an HPLC analytical method was developed to determine the content of four chromones in 20 batches of SR samples based on the CRE with known content as the standard substance. T-test was used for the comparison of the determination results of the two methods(single chemical component and CRE as reference substances, respectively), and the P values of prim-O-glucosylcimifugin, cimifugin, 4'-O-ß-D-glucosyl-5-O-methylvisamminol, and sec-O-glucosylhamaudol were 0. 16,0. 39, 0. 14, and 0. 42. The results demonstrated that there was no significant difference between the two methods. This study initially verified the feasibility that the CRE could be used as a substitute for single reference substance in quality control of SR. In conclusion,this study is expected to provide a scientific basis and a new research model for the application of reference extract in the quality control of Chinese medicine.

[Study on scrophulariaceae radix reference extract and its application in quality control of Scrophulariae Radix].

By establishing the preparation process of Scrophulariaceae Radix reference extract(SRRE) and calibrating it, we discussed its feasibility as a substitute for single reference substance in the quality control of Scrophulariae Radix. The SRREs were prepared by solvent extraction method and chromatographic separation technology, and then calibrated with the reference substances of harpagide, angoroside C and harpagoside. The HPLC content determination method of Scrophulariae Radixl was established with SRREs of the known content and the reference substances of harpagide, angoroside C and harpagoside respectively as the control ones. Then the content of three components in Scrophulariae Radix was determined, and the t-test method was used to compare the results of the two methods. With SRRE as references, harpagide, angoroside C and harpagoside were in a good linear relationship(r≥0.999 8) within each range, and the average recovery rate was 98.55% to 100.6%. The t-test results showed that the P values of two determination methods were 0.493, 0.155 and 0.171 for harpagide, angoroside C and harpagoside respectively, indicating no significant diffe-rence between the two methods of content determination. The SRRE can be used as a substitute for the reference in the quality control of Scrophulariaceae Radix. The SRRE can replace the corresponding reference substance for the quality control of Scrophulariae Radix. The results of this study provide new methods and new ideas for the quality evaluation of Scrophulariae Radix, and provide a scientific basis for the application of reference extracts in the quality research of traditional Chinese medicine.

[Construction of "2R network pharmacology" research method for effective components of traditional Chinese medicine based on "prediction of dominant components-screening of potential targets"].

Chinese traditional medicine compound is the main form of Chinese medicine clinical application. The elucidation of the effective components of traditional Chinese medicine is one of the key scientific issues to promote the modernization of traditional Chinese medicine. At present, there are many research ideas on the effective components of traditional Chinese medicine compounds. By analyzing the current status and existing problems of existing research ideas, the author proposes a "double reduction network pharmacology"(2 R network pharmacology) research method based on "prediction of dominant components-potential target selection". Chemical components with good properties were selected by ADMET property prediction technology, and compared with the blood components and target organ components to determine the dominant components with potential therapeutic effect, that is "reducing constituents"; the potential core regulatory pathway of traditional Chinese medicine compound was enriched by RNA-Seq technology combined with network database, and then the target of traditional Chinese medicine compound was mined based on the signal pathway, that is "reducing targets". To improve the efficiency and accuracy of effective component screening, the network relationship of "component target" was established by the related technology of network pharmacology. The purpose of this study is to provide practical research ideas and methods for clarifying the effective components of traditional Chinese medicine, revealing the law of compatibility of traditional Chinese medicine and clarifying the target of drug action.

[Identification of compounds in Lycii Cortex by UPLC-LTQ-Orbitrap-MS].

This experiment was performed to analyze and identify the chemical constituents of Lycii Cortex by UPLC-LTQ-OrbitrapMS. The analysis was performed on a Waters Xbridge Shield RP18( 4. 6 mm×250 mm,5 µm) column with the mobile phase of 0. 1%formic acid( A)-acetonitrile( B) under gradient conditions at a flow rate of 1. 0 m L·min-1 and the temperature maintained at 35 ℃ ．Electrospray ionization ion trap time-off light multistage mass spectrometry was applied for qualitative analysis under positive and negative ion modes. The results indicated that 55 compounds consisted of 39 phenolic amides,6 organic acids,3 cyclic peptides,2 coumarins and 5 others. In conclusion,an UPLC-LTQ-Orbitrap-MS method was established to qualitative analysis of Lycii Cortex in this study,and the fragmentation rules of phenolic amides were summarized,which provides a good foundation for further study of Lycii Cortex.

Tissue engineered esophagus by mesenchymal stem cell seeding for esophageal repair in a canine model.

PURPOSE: Acellular porcine small intestinal submucosa (SIS) has been successfully used for esophagoplasty in dogs. However, this has not led to complete epithelialization and muscular regeneration. We undertook the present study to assess the effect of tissue-engineered esophagus generated by seeding bone marrow mesenchymal stem cells (BMSCs) onto an SIS scaffold (BMSCs-SIS) in a canine model. METHODS: We cultured, passaged, and measured autologous BMSCs and myoblasts with cell proliferation and immunohistochemical assays. We labeled the third passage of BMSCs with PKH-26, a fluorescent dye, before seeded it onto the SIS. We resected canine cervical esophagus to generate a defect 5 cm in length and 50% in circumference, which we repaired with BMSCs-SIS or SIS alone. RESULTS: Four weeks later, barium esophagram demonstrated that esophageal lumen surface of the patch graft was smoother in the BMSCs-SIS group compared with the SIS group. Histological examination suggested a strong similarity between BMSCs and esophageal myoblasts in terms of morphology and function. Although both BMSCs-SIS and SIS repaired the esophageal defects, we noted complete re-epithelialization with almost no inflammation only in the former group. By 12 wk after the surgery, we observed long bundles of skeletal muscles only in the BMSCs-SIS group, where the microvessel density was also much greater. CONCLUSIONS: Bone marrow mesenchymal stem cells on an SIS scaffold can promote re-epithelialization, revascularization, and muscular regeneration. This approach may provide an attractive option for esophageal regeneration.

Hypoxia inhibits the spontaneous calcification of bone marrow-derived mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are the popular seed cells for regenerative medicine, and there has been a rapid increase in the number of BM-MSC-based clinical trials. However, the safety of these cells should also be closely studied. In this study, spontaneous calcification of BM-MSCs from rats was evaluated in normoxia (20% O(2)) without osteogenic medium after continuous culture for 21 days; obvious mineralized nodules were observed, which were positive for Alizarin Red, collagen-I (Col-I), osteocalcin (OC) and alkaline phosphatase (ALP), and mainly consisted of C, O and Ca elements. Interestingly, hypoxia (2% O(2)) significantly inhibited this spontaneous calcification. In addition, the ALP and calcium content of rBM-MSCs were sharply reduced. Based on RT-PCR results, the expression of osteogenic genes (Cbfa1/Runx2, Col-I, ALP, and OC) was reduced compared to that in normoxia. These results demonstrate a natural and unique characterization of rat BM-MSCs in normoxia after continuous culture and highlight the inhibiting effects of hypoxia. Finally, this study contributes to the information regarding the application of BM-MSCs in the regeneration of various tissues.

Sonic hedgehog enhances the proliferation and osteogenic differentiation of bone marrow-derived mesenchymal stem cells.

MSCs (mesenchymal stem cells) may be promising seed cells for tissue regeneration because of their self-renewal and multi-differentiation potential. Shh (sonic hedgehog) is involved in the skeletal formation during embryo development and skeletal regeneration. However, how Shh regulates the biological characteristics of BM-MSCs (bone marrow-derived MSCs) is poorly understood. We have investigated the effect of rShh-N (recombinant N-terminal Shh) on the proliferation and osteogenic differentiation of rBM-MSCs (rat BM-MSCs) in vitro. rBM-MSCs were treated with rShh-N at concentrations up to 200 ng/ml. Proliferation and colony-forming ability of rBM-MSCs were increased in a dose-dependent manner. rShh-N increased the ratio of cells in S and G2/M phase, as well as the number of Ki-67+ cells. In addition, ALP (alkaline phosphatase) activity and matrix mineralization were enhanced by 200 ng/ml rShh-N. Real-time PCR showed that rShh-N (200 ng/ml) up-regulated the expression of genes encoding Cbfa-1 (core-binding factor α1), osteocalcin, ALP and collagen type I in rBM-MSCs. This information reveals some potential of rShh-N in the therapeutics of bone-related diseases.

Deciphering the effective combinatorial components from Si-Miao-Yong-An decoction regarding the intervention on myocardial hypertrophy.

ETHNOPHARMACOLOGICAL RELEVANCE: Si-Miao-Yong-An decoction (SMYAD), a classical traditional Chinese medicine (TCM) formula, has been used to treat various cardiovascular diseases in clinics. AIM OF THE STUDY: The aim of this study is to investigate the effective combinatorial components from SMYAD and its mechanism regarding the intervention on myocardial hypertrophy. MATERIALS AND METHODS: SMYAD constituents absorbed in rat plasma and heart were identified using UHPLC Q-Exactive-Orbitrap MS/MS. The identified constituents in SMYAD were further analyzed using ADMET (absorption, distribution, metabolism, excretion and toxicity) prediction and molecular docking. The effective constituents were identified using isoproterenol (ISO)-induced H9c2 cardiomyocyte hypertrophy, and neochlorogenic acid (NCA), chlorogenic acid (CA), cryptochlorogenic acid (CCA), isochlorogenic acid C (ICAC), angoroside C (AGDC), isochlorogenic acid A (ICAA), sweroside (SRD), and harpagide (HPD) in SMYAD extract were quantified by HPLC for compatibility. Finally, anti-hypertrophic activities of candidate effective combinatorial components, which were prepared according to the determined molar concentration ratio of effective constituents using reference substance solution, were analyzed using immunofluorescence staining and Quantitative real-time PCR. The expression levels of PI3Kα, p-ERK, p-Akt, Akt, p-mTOR, mTOR and HIF-1α were measured using Western blot. RESULTS: 32 prototypes of SMYAD were identified from plasma and heart tissue of rat. Combining with ADMET prediction, 31 dominant constituents were focused. Based on HIF-1 pathway identified in preliminary result, 17 targets were focused, which were used to dock with 31 constituents. 27 constituents were therefore hit as the potential effective constituents of SMYAD in inhibiting myocardial hypertrophy. Bioactivity evaluation showed that NCA, CA, CCA, ICAC, AGDC, ICAA, SRD, and HPD significantly inhibited the increase of H9c2 cell surface area induced by ISO. Except for ICAA and AGDC, the remaining 6 effective constituents, showing a certain inhibitory effect on ISO-induced ANP mRNA overexpression at high and low concentrations, participated in compatibility based on the molar concentration ratio determined by HPLC. Effective combinatorial components composed of the 6 effective constituents (effective combinatorial components ABC) showed significant inhibitory effect on the increase of cell surface area, and the overexpression of ANP and ß-MHC mRNA in H9c2 cells induced by ISO. Moreover, effective combinatorial components ABC significantly inhibited the protein overexpressions of p-Akt, p-mTOR and HIF-1α. Based on the results, we put forward the strategy of "Focusing constituents" and "Focusing targets" for the effective constituents research of TCM formula. CONCLUSION: Effective combinatorial components ABC composed of NCA, CA, CCA, ICAC, SRD and HPD from SMYAD inhibited ISO-induced cardiomyocyte hypertrophy and down-regulated expression of ANP and ß-MHC mRNA through the inactivation of Akt/mTOR/HIF-1α pathway.

Characterization of MSCs from human placental decidua basalis in hypoxia and serum deprivation.

Recently, we reported that human PDB (placental decidua basalis) is an excellent source of MSCs (mesenchymal stem cells), meanwhile, PDB-MSCs could survive under hypoxia and serum deprivation. Herein, we investigated the proliferation, clonogentic efficiency, phenotypes, metabolic activity and cytokines secretion of PDB-MSCs in hypoxia and serum deprivation. PDB-MSCs were cultured in four groups: normoxia (20% O2) and complete medium [10% FBS (foetal bovine serum)+DMEM-HG (Dulbecco's modified Eagle's medium-high glucose)], hypoxia and complete medium, normoxia and serum deprivation (0% FBS), and hypoxia and serum deprivation. After 96 h of culture in the above groups, PDB-MSCs maintain the phenotypes stably. Interestingly, hypoxia notably enhanced the proliferation, colony-forming potential and lactate/glucose ratio in complete medium, but suppressed the secretion of BMP-2 (bone morphogenetic protein-2) and bFGF (basic fibroblast growth factor), while it did not change the quantity of VEGF (vascular endothelial growth factor) and bFGF in serum deprivation. Although PDB-MSCs grew slowly and seldom formed a colony unit in hypoxia and serum deprivation, they possessed a moderate metabolism. In conclusion, our results indicate that PDB-MSCs appear to be promising seed cells for ischaemia-related tissue engineering.

Grafts of porcine small intestinal submucosa with cultured autologous oral mucosal epithelial cells for esophageal repair in a canine model.

The acellular porcine small intestinal submucosa (SIS) has been successfully used for esophagoplasty. However, it does not lead to a complete epithelialization in a canine model. A cellular component may be required for better reconstruction. The present study was undertaken to investigate the feasibility and effectiveness of the combination of SIS and autologous oral mucosal epithelial cells (OMECs) for esophageal reconstruction. The OMECs harvested from beagle dogs were cultured and propagated, and the 3rd passage cells were seeded on a single-layer SIS. Male beagle dogs were subjected to surgical resection to produce cervical esophageal defects (5 cm in length, 180 degrees in range). SIS with or without OMECs was patched on the esophageal defects. Barium esophagram, immunohistochemistry, and histology were performed to evaluate the therapeutic effectiveness. Four weeks after surgery, the histological examination showed a complete re-epithelialization and almost no inflammation in the SIS with OMECs group. But in the SIS group, only a partial epithelialization was observed along with inflammation. Eight weeks after surgery, the squamous epithelium was found to cover the entire graft surface in both groups; however, the muscular regeneration was observed in the SIS with OMECs, but not in the SIS group. The graft of SIS combined with autologous OMECs promotes re-epithelialization and muscular regeneration. It is an effective alternative method for esophageal repair.

[Effects of hypoxia on human placental decidua basalis-mesenchymal stem cells proliferation, apoptosis and VEGF expression.].

Human placental decidua basalis-mesenchymal stem cells (PDB-MSCs) are multipotent cells from the human term placenta, which are ethically conducive, easily accessible and high-yielding source. PDB-MSCs can differentiate into adipogenic, osteogenic and neurogenic cells under appropriate conditions, which may be an attractive and alternative source of seed cells for tissue engineering. To investigate the effect of hypoxia (1% O2) on human PDB-MSCs and the expression of cytokine, PDB-MSCs were isolated from human placenta by density gradient centrifugation and cultured in the Dulbecco's modified Eagle's medium-high glucose (DMEM-HG) containing 10% fetal bovine serum (FBS), and the fifth passage of PDB-MSCs were taken. PDB-MSCs were divided into 4 groups according to the concentrations of O2 and FBS: 20% O2, 10% FBS; 20% O2, 0% FBS; 1% O2, 10% FBS; 1% O2, 0% FBS. The proliferation and apoptosis of PDB-MSCs were detected by MTT and flow cytometric analysis at the time points of 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h, respectively. Vascular endothelial growth factor (VEGF) released from PDB-MSCs was detected by enzyme-linked immunosorbent assay (ELISA) at the same time points. The results showed that hypoxia enhanced the proliferation of PDB-MSCs at 12 h under the condition of 10% FBS, while at 24 h under the condition of 0% FBS (P<0.01, n=3). In normoxia, the cells cultured in 10% FBS displayed a significant proliferation compared to those cultured in 0% FBS. However, in hypoxia, the number of cells cultured in 0% FBS (serum deprivation) increased significantly compared to that cultured in 10% FBS at 24 h and 96 h respectively (P<0.05, P<0.01, n=3). With the flow cytometric analysis of cell apoptosis under the condition of hypoxia and serum deprivation, we found that hypoxia and serum deprivation did not induce PDB-MSCs apoptosis (P>0.05, n=3). This conclusion may relate to the expression of VEGF which needs further research. In conclusion, the results obtained indicate that PDB-MSCs are able to bear hypoxia and serum deprivation, suggesting that PDB-MSCs can be used as seed cells for ischemia related tissue engineering.

[An experimental study on targeting suicide gene therapy for lung cancer with HSV-TK driven by hTERT promoter].

OBJECTIVE: To study the approach of targeting expression of suicide gene HSV-TK driven by human telomerase catalytic subunit (hTERT) promoter in lung cancer cells, and to investigate inhibitory effect of HSV-TK/GCV driven by hTERT promoter on proliferation of lung cancer cell line A549 in vitro and in vivo. METHODS: (1) Recombinant expression vectors of HSV-TK driven by hTERT promoter and SV40 promoter (pGL3-hTp-TK and pGL3-SV40-TK) were transfected into telomerase-positive human lung adenocarcinoma cell A549 and telomerase-negative human embryonic lung fibroblast cell MRC-5. The mRNA expression of TK gene was detected with RT-PCR method; (2) With the treatment of GCV, the proliferation of above transfected cells was investigated by MTT assay; Influence of GCV on apoptosis and cell cycle of these cells was evaluated with flow cytometry; (3) After the subcutaneously transplantation of A549 cells into nude mice, intra-tumor injection of plasmid-liposome as well as intra-peritoneal injection of GCV were performed to stUdy anti-tumor effects of pGL3-hTp-TK/GCV and pGL3-SV40-TK/GCV in vivo. RESULTS: (1) Enzyme digestion and PCR suggested that recombinant plasmids of pGL3-hTp-TK and pGL3-SV40-TK were successfully constructed; TK mRNA expression was detected in both A549 and MRC-5 cells transfected with pGL3-SV40-TK, also in A549 transfected with pGL3-hTp-TK, but not in MRC-5 transfected with pGL3-hTp-TK; (2) GCV showed significant inhibition effect on proliferation of A549 and MRC-5 transfected with pGL3-SV40-TK in vitro, also on that of A549 transfected with pGL3-hTp-TK, but not of MRC-5 transfected with pGL3-hTp-TK; With the treatment of GCV, apoptosis index (AI) of A549 cells transfected with pGL3-SV40-TK and pGL3-hTp-TK (21.58% and 23.19% respectively) increased significantly, compared with that of A549 transfected with pGL3-hTp and blank control; GCV enhanced the effects on AI in MRC-5 transfected with pGL3-SV40-TK (9.35%), but not with pGL3-hTp-TK (0.88%); (3) Inhibition ratio of pGL3-SV40-TK/GCV and pGL3-hTp-TK/GCV to transplanted tumor of A549 in nude mice (46.7% and 40.5% respectively) were significantly higher than that of negative control groups (9.7%, 14.3%, 7.0% and 8.0% respectively). CONCLUSION: TK gene driven by hTERT promoter could express selectively in lung cancer cell. Lung cancer cell could be specifically inhibited by HSV-TK/GCV driven by hTERT promoter in vitro and in vivo.

[Stably transfection of DNA to human fibroblasts: a comparative study of three approaches].

OBJECTIVE: To search for the best approaches to transfect plasmid pEGFP-N1 into human fibroblasts. METHODS: Cationic polymer mediated transfection, cationic liposome transfection and electrotransfection were compared for transfecting plasmid pEGFP-N1, a plasmid that expresses G418 resistance protein and Enhanced green fuluorescent protein (EGFP), into Human fibroblasts. The expression of EGFP was observed 24 hours after the transfection by fluorescence microscope. The transient transfection efficiency was measured by flow cytometry. The stable transfected cells were screened by G418. RESULTS: Electrotransfection had the highest transient transfection efficiency (56.8%), with the most masc clones of stably transfection and the least time taken to form big masc clones (20 days). Cationic polymer mediated transient transfection had the lowest efficiency(1.7%), with the least masc clones and the longest time taken to form the big masc clones (30 days). Cationic liposome transfection had a transient transfection efficiency of 39.9% and took 25 days to form the big masc clones. CONCLUSION: Electrotransfection is the best way of stably transfecting plasmid pEGFP-N1 into human fibroblasts.

[A study on selective killing effect of Hsv-tk/GCV driven by human telomerase catalytic subunit promoter on human lung cancer cell A549].

OBJECTIVE: To study selective killing effect of herpes simplex virus-thymidine kinase/ganciclovir (Hsv-tk/GCV) driven by human telomerase catalytic subunit (hTERT) promoter on lung cancer cell line A549 in vitro. METHODS: (1) Expression plasmids of Hsv-tk gene driven by hTERT promoter and sv40 promoter respectively (pGL3-hTp-tk and pGL3-sv40-tk) were transfected into telomerase-positive human lung adenocarcinoma cell line A549 and telomerase-negative fetal lung fibroblast cell line MRC-5. Reverse transcription-PCR was performed to detect expression of tk gene in above transfected cell lines; (2) Inhibition effect on proliferation of above transfected cell lines treated with GCV was investigated with MTT method; (3) Influence of GCV on apoptosis and cell cycle of above transfected cell lines was investigated with flow cytometry. RESULTS: (1) tk mRNA expression was detected in both A549 and MRC-5 transfected with pGL3-sv40-tk, also in A549 transfected with pGL3-hTp-tk, but not in pGL3-hTp-tk transfected MRC-5; (2) GCV showed significant inhibition effects on proliferation of pGL3-sv40-tk transfected A549 and MRC-5 in vitro, also on that of pGL3-hTp-tk transfected A549, but not on that of pGL3-hTp-tk transfected MRC-5; (3) Treated with GCV, apoptosis index (AI) of pGL3-sv40-tk transfected A549 and MRC-5 as well as pGL3-hTp-tk transfected A549 (21.58%, 9.35% and 23.19% respectively) increased significantly, compared with A549, MRC-5 transfected with pGL3-hTp (0.78% and 0.55% respectively) and A549, MRC-5 without plasmid transfection as blank control (2.17% and 0.60% respectively); GCV had no influence on AI of pGL3-hTp-tk transfected MRC-5 (0.88%). CONCLUSION: tk gene driven by hTERT promoter could express selectively in lung cancer cell A549. Hsv-tk/GCV driven by hTERT promoter could selectively inhibit proliferation of lung cancer cell.

Replication of IGF2-INS-TH*5 haplotype effect on obesity in older men and study of related phenotypes.

Interindividual variation of the IGF2-INS-TH region influences risk of a variety of diseases and complex traits. Previous studies identified a haplotype (designated IGF2-INS-TH(*)5 and tagged by allele A of IGF2 ApaI, allele 9 of TH01 and class I alleles of INS VNTR) associated with low body mass index (BMI) in a cohort of UK men. We aimed here both to study whether previous findings relating (*)5 with weight are replicated in a different cohort of men (East Hertfordshire) characterised in more phenotypic detail and to test the effect of this haplotype on related subphenotypes. The PHASE program was used to identify (*)5 and not(*)5 haplotypes. A total of 490 haplotypes were derived from 131 men and 114 women, the frequency of (*)5 being around 9%. Specific tests of (*)5 haplotype (vs not(*)5 haplotypes) conducted included Student's t-test and multiple regression analyses. We observed replication of weight effect for the (*)5 haplotype in men: significant associations with lower BMI (-1.81 kg/m(2), P=0.009), lower waist circumference (-6.3 cm, P=0.001) and lower waist-hip ratio (-5%, P<0.001). This haplotype also marks nearly two-fold lower 120 min insulin (P=0.004) as well as low baseline insulin (-11.02 pmol/l, P=0.043) and low 30 min insulin (-64.44 pmol/l, P=0.072) in a glucose tolerance test. No association between (*)5 and these traits was found in women. Our results, taken together with other data on IGFII levels and TH activity, point to the importance of (*)5 as an integrated polygenic haplotype relevant to obesity and insulin response to glucose in men.

[Study on cloning of hTERT promoter and its targeting transcriptional activities in telomerase-positive lung cancer cells].

OBJECTIVE: To clone sequence of hTERT promoter and study its transcriptional activity and its relationship with hTERT mRNA expression and telomerase activity in various kinds of human lung cancer cells and normal cells, and to investigate the targeting transcriptional activity of hTERT promoter in tumor cells. METHODS: About 1.1 kb promoter of the 5'flanking sequence of the hTERT was amplified from genomic DNA isolated from 293 cells by polymerase chain reaction (PCR). After being confirmed by DNA sequencing, the hTERT promoter was inserted into luciferase reporter vectors (pGL3-basic) to reconstruct a recombinant named pGL3-hTERTp. Then pGL3-hTERTp was transiently transfected into lung cancer cell A549, SPC-A-1, LTEPa-2, NCI-H446, YTMLC-9, GLC-82, 95D, A2, and normal cell of MRC-5. The transcriptional activities of hTERT promoter in various cells were determined by measuring the luciferase activities. hTERT mRNA expression and telomerase activity were determined by RT-PCR and TRAP ELISA. RESULTS: Eelectrophoresis demonstrated that the hTERT promoter amplified by PCR was about 1.1 kb long, and DNA sequencing showed a sequence the same as the hTERT promoter registered in GenBank being 1084 bp in length. The recombinant of plasmid pGL3-hTERTp was confirmed by double digestion and PCR methods with correct results. hTERT mRNA and telomerase activity were expressed in all of eight lung cancer cell lines at varied levels, but not expressed in normal cell. Transient transfection assay and Luciferase assay also revealed that hTERT promoter had different transcriptional activities in various lung cancer cells, but no transcriptional activity was shown in normal cells. CONCLUSION: 1084 bp hTERT promoter cloned has specific transcriptional activities in various telomerase-positive lung cancer cells, and it may act as control element in tumor-targeting gene therapy.

Late life metabolic syndrome, early growth, and common polymorphism in the growth hormone and placental lactogen gene cluster.

Low rates of fetal and infant growth are associated with the metabolic syndrome and cardiovascular disease in later life. We investigated common genetic variation in the GH-CSH gene cluster on chromosome 17q23 encoding GH, placental lactogens [chorionic somatomammotropins (CSH)], and placental GH variant in relation to fetal and infant growth and phenotypic features of the metabolic syndrome in subjects aged 59-72 yr from Hertfordshire, UK. Allele groups T, D1, and D2 of a locus herein designated CSH1.01 were examined in relation to GH-CSH single nucleotide polymorphisms and to specific phenotypes. Average birth weights were similar for all genotype groups. Men with T alleles were significantly lighter at 1 yr of age, shorter as adults, and had higher blood pressures, fasting insulin (T/T 66% higher than D2/D2) and triglyceride concentrations, and insulin and glucose concentrations during a glucose tolerance test. Birth weight and 1-yr weight associations with metabolic syndrome traits were independent of the CSH1.01 effects. Common diversity in GH-CSH correlates with low 1-yr weight and with features of the metabolic syndrome in later life. GH-CSH genotype adds substantially to, but does not account for, the associations between low body weight, at birth and in infancy, and the metabolic syndrome.

Microplate array diagonal gel electrophoresis for cohort studies of microsatellite loci.

After PCR amplification, we have achieved precise sizing of trinucleotide and tetranucleotide microsatellite alleles on 96-well open-faced polyacrylamide microplate array diagonal gel electrophoresis (MADGE) gels: two tetranucleotide repeats, HUMTHOI (five alleles 248-263 bp) and DYS390 (eight alleles 200-228 bp), and DYS392, a trinucleotide repeat (eight alleles 210-231 bp). A gel matrix of Duracryl, a high mechanical strength polyacrylamide derivative, and appropriate ionic conditions provide the 1.3%-1.5% band resolution required. No end-labeling of primers is needed, as the sensitive Vistra Green intercalating dye is used for the visualization of bands. Co-run markers bracketing the PCR fragments ensure accurate sizing without inter-lane variability. Electrophoresis of multiple gels in a thermostatically controlled tank allows up to 1000 samples to be run in 90 min. Gel images were analyzed using a Fluorlmager 595 fluorescent scanning system, and alleles were identified using Phoretix software for band migration measurement and Microsoft Excel to compute fragment sizes. Estimated sizes were interpolated precisely to achieve accurate binning. Microsatellite-MADGE represents a utilitarian methodfor high-throughput genotyping in cohort studies, using standard laboratory equipment.

[A study of the inhibition effect of HIFU and its mechanism of action on the proliferation of human breast cancer cells].

OBJECTIVE: To investigate the killing effect of high intensity focused ultrasound (HIFU) on human tumor cells and study the mechanism of its action. METHODS: Human breast cancer cells MCF-7 were treated with different intensity of HIFU in vitro and incubated. The killing effect and proliferation inhibition effect of HIFU on tumor cells were investigated with the methods of trypan blue exclusion assay, MTT assay and colony formation assay. And the cell cycle, apoptotic cells and the expressions of P53, BCL-2, FAS and HSP70 were measured with flow cytometry. RESULTS: After HIFU treatment, the death rates of cancer cell increased, the proliferation of cell waned (P < 0.05), the number of colony formation decreased (P < 0.001), the cell number increased in G0/G1 phase (P < 0.05) and decreased in S phase, the apoptotic indices increased (P < 0.01), the expression of HSP70 was enhanced, and no significant changes of P53, BCL-2, FAS expressions were found (P > 0.05). CONCLUSION: HIFU produces significant effects such as killing, proliferation inhibition and colony formation inhibition on human breast cancer cells; its mechanism of action may be associated with the inhibition of DNA synthesis. In addition, HIFU treatment can induce apoptotic cell death, which may not be associated with the mediation and regulation of P53, BCL-2 and FAS genes.

[Study on revising the criteria of classification of hazard conditions of productive dust].

OBJECTIVE: To provide scientific basis data for revising the national hygiene criteria of "Classification of hazard conditions of productive dust" (GB5817-86). METHODS: The data of the retrospective study and the field survey data were analyzed with correlation and regression analysis. The product of total dust concentration of respiratory exposure (mg/m(3)), total ventilation during exposure (m(3)/d per psrson), and level of free SiO(2) in dust (%) was the respiratory exposure dose of free SiO(2) (mg per day per person) which was used as dose criteria value of classification of hazard degree of dust. RESULTS: Using free SiO(2) exposure dose and the dose-effect relationship, the hazard degrees of the dust were divided into 5 grades: 0, I, II, III, IV (0 - 8.0, 8.1 - 12.0, 12.1 - 16.0, > 24.1 mg per day per person). CONCLUSION: The exposure dose of free SiO(2) is closely related to the pathogenesis of silicosis. Using the exposure dose of free SiO(2) as the classification indicator of hazard degree of dust is reliable, simple and easy to execute.