This study was aimed to establish the thin layer chromatography (TLC) identification and content determination of Tibetan medicineJi-Ni De-Xie (JNDX). TLC was used to identifyBerberis kansuensisSchneid,Terminalia chebula Retz.,Phyllanthus emblicaandCurcuma LongaL. The high performance liquid chromatography (HPLC) was used to determine the content of berberine hydrochloride. The results showed that the TLC identification had a good resolution with clear spots and no interference from the negative reference. The content of berberine hydrochloride showed a good linear relationship with the peak area in the range of 0.05-0.45 mg·mL-1. The regression equation wasY = 40 679X-130.56. The average recovery was 99.07%. It was concluded that the established method was easy to operate with good repeatability. It can effectively control the quality of Tibetan medicine JNDX.

OBJECTIVETo explore the molecular mechanisms potentially responsible for carcinogenesis due to cadmium by detecting expression change of the translation initiation factor 3 (TIF3 p36) in those malignant transformation of human bronchial epithelial cell lines (16HBE) induced by cadmium chloride (CdCl(2)).

METHODSThe expression changes of TIF3 p36 were detected and analyzed at different stages of malignant cells (semi transformed cells, transformed cells and tumorigenic cells) induced by CdCl(2) solution with both reverse transcription PCR technique and sensitive fluorescent quantitative PCR assay.

RESULTSCompared with non-transformed human bronchial epithelial cells, the results of fluorescent quantitative PCR assay showed that the semi-transformed cells, transformed cells and tumorigenic cells all expressed higher levels of TIF3 p36 mRNA (P < 0.01 or P < 0.05). As compared with the control cells, the TIF3 expressions at different stages of malignant transformation were 3.1 times, 5.9 times and 9.9 times higher respectively in the low dosage group of CdCl(2) (5 micromol/L); 7.1 times, 6.8 times and 14.8 times respectively in the middle dosage group of CdCl(2) (10 micromol/L); 3.6 times, 3.0 times and 9.1 times respectively in high of dose of CdCl(2) (15 micromol/L). These results showed that there was the positive correlation between overexpression levels of TIF3 p36 mRNA and the malignant degree of the cells, but they were not related to the dosages of cadmium.

CONCLUSIONThere is significantly abnormal overexpression of TIF3 gene during malignant transformation of human bronchial epithelial cell line induced by cadmium chloride, and the TIF3 expression is associated with the malignant degree of the cells, which may be one of molecular mechanisms potentially responsible for the carcinogenesis due to cadmium.

Bronchi ; cytology ; Cadmium Chloride ; toxicity ; Cell Line ; Cell Line, Transformed ; Cell Transformation, Neoplastic ; chemically induced ; metabolism ; Dose-Response Relationship, Drug ; Humans ; Prokaryotic Initiation Factor-3 ; biosynthesis ; genetics ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo study the alternative expression and sequence of human elongation factor-1 delta (human EF-1 delta p31) during malignant transformation of human bronchial epithelial cells induced by cadmium chloride (CdC12) and its possible mechanism.

METHODSTotal RNA was isolated at different stages of transformed human bronchial epithelial cells (16HBE) induced by CdCl2 at a concentration of 5.0 microM. Special primers and probe for human EF-1 delta p31 were designed and expression of human EF-1 delta mRNA from different cell lines was detected with fluorescent quantitative PCR technique. EF-18 cDNA from different cell lines was purified and cloned into pMD 18-T vector followed by confirming and sequencing analysis.

RESULTSThe expressions of human EF-1 beta p31 at different stages of 16HBE cells transformed by CdCl2 was elevated (P < 0.01 or P < 0.05). Compared with their corresponding non-transformed cells, the overexpression level of EF-1 delta p31 was averagely increased 2.9 folds in Cd-pretransformed cells, 4.3 folds in Cd-transformed cells and 7.2 folds in Cd-tumorigenic cells. No change was found n the sequence of overexpressed EF-1beta p31 at different stages of 16HBE cells transformed by CdCl2.

CONCLUSIONOverexpression of human EF-1beta p31 is positively correlated with malignant transformation of 16HBE cells induced by CdC12, but is not correlated with DNA mutations.

Cadmium Chloride ; Cell Line ; Cell Transformation, Neoplastic ; chemically induced ; metabolism ; Epithelial Cells ; drug effects ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; Humans ; Peptide Elongation Factor 1 ; genetics ; metabolism ; Respiratory Mucosa ; drug effects ; metabolism ; pathology ; Sequence Analysis, DNA

OBJECTIVETo analyze the relationship between malignant transformation and abnormal expression of eukaryotic initiation factor 3 (eIF3 p36) in human bronchial epithelial (16HBE) cells induced by cadmium chloride (CdCl2).

METHODS16HBE cells were treated several times with different concentrations of CdCl2. Tumorigenic potential of transformed cells was identified by assays for anchorage-independent growth in soft agar and for tumorigenicity in nude mice after the 35th passage. Total RNA was isolated from 16HBE cells induced by CdCl2, including non-transformed, Cd-transformed, and Cd-tumorigenic cell lines. Special primers for eIF3 p36 were designed and the expression of eIF3 mRNA in different cell lines was detected with fluorescent quantitative-polymerase chain reaction technique (FQ-PCR).

RESULTSThe 35th passage of 16HBE cells transformed by CdCl2 exhibited overlapping growth. Compared with the non-transformed cells, colonies of transformed cell lines in soft agar showed statistically significant increases and dose-dependent effects (P<0.01). All Cd-induced transformed cell lines formed tumors in nude mice within 2 weeks of inoculation, but none of the mice injected with non-transformed cells showed tumors even after 3 weeks. All tumors were pathologically identified as poorly differentiated squamous cell carcinoma. The eIF3 p36 genes in different stages of 16HBE cells transformed by CdCl2 were elevated as compared with the non-transformed control (P<0.01), and the eIF3 expression increased with the degree of cell malignancy.

CONCLUSIONCdCl2 is capable of inducing morphological transformation in 16HBE cells and transformed cells are potentially tumorigenic. Over-expression of eIF3 p36 is positively correlated with malignant transformation of 16HBE cells induced by CdCl2 and may be one of the molecular mechanisms potentially responsible for carcinogenesis due to Cd.

Animals ; Base Sequence ; Bronchi ; cytology ; drug effects ; metabolism ; Cadmium Chloride ; pharmacology ; Cell Transformation, Neoplastic ; DNA Primers ; Epithelial Cells ; drug effects ; metabolism ; Eukaryotic Initiation Factors ; metabolism ; Humans ; Mice ; Mice, Nude ; Polymerase Chain Reaction

OBJECTIVETo observe the short-term clinical results of the adjacent segment degeneration after the implantation of Coflex system at the interspinous space of adjacent segment to lumbar fusion.

METHODSFifty patients with grade III disc (Thompson MRI classification) of adjacent segment to lumbar fusion were included and divided alternately into two groups according to the order of hospitalization from January to November 2009. Coflex system was implanted at the interspinous space of adjacent segment to lumbar fusion in 25 patients as Coflex group, the other 25 patients did not have any surgical treatment were as control group. The followed up time was 2 years. Visual analogue scale (VAS) score of low back pain, changes of disc height and motion range of adjacent segment to lumbar fusion on X-ray imaging were evaluated by independent sample t-test or paired samples t-test.

RESULTSThere were 22 patients in Coflex group and 21 patients in control group were followed up 2 years post-operation. The difference of VAS score between two groups was no significance (P > 0.05). In Coflex group, the change of postoperative disc height was no significance (P > 0.05), but the motion range was significantly reduced to 47% of the preoperative value (t = 7.99, P < 0.05). In control group, the postoperative disc height decreased slightly, without significant difference to the preoperative value (P > 0.05). Between the two groups, no differences of the disc height and motion range were found before operation, but the differences of the disc height changes (t = 6.7, P < 0.05) and motion rang (t = -14.5, P < 0.05) were significant in 2 years post-operation. No complications such as Coflex system loosen, immigration and spinal process fracture were occurred.

CONCLUSIONSCoflex system can obviously limit the motion range and maintain the disc space height of adjacent segment to lumbar fusion, and prevent its degeneration in some degree.

Adult ; Female ; Follow-Up Studies ; Humans ; Internal Fixators ; Lumbar Vertebrae ; surgery ; Male ; Middle Aged ; Postoperative Complications ; prevention & control ; Prospective Studies ; Spinal Fusion ; adverse effects ; instrumentation ; methods ; Treatment Outcome

OBJECTIVETo explore the expression and sequence of ERCC1 gene in CdCl2-induced transformed human bronchial epithelial 16HBE cells at different stages.

METHODSReverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemcial staining (SP method) were used to measure the ERCC1 mRNA and protein expression in 16HBE cells at different passages treated with CdCl2 (the 5th, 15th, 35th passage, and neoplastic cells from tumors formed in nude mice). ERCC1 exon 3,exon 4 of the 16HBE cells and tumor cells from nude mice were amplified by polymerase chain reactions (PCR), the amplified DNA strips were purified,and the exons were detected by DNA analysis.

RESULTSDuring the passages of 16HBE cells treated with CdCl2, the expression of ERCC1 gene was decreased gradually. The ERCC1 gene mRNA and protein expression levels of the CdCl2-transformed 35th passage 16HBE cells and tumor cells from nude mice were significantly decreased comparing with those in non-transformed 16HBE cells (P < 0.01). In the CdCl2-induced tumorigenic cells in nude mice, there was adenine (A) deletion in 1st site of ERCC1 exon 4. The mutation was frame shift mutation.

CONCLUSIONThe decreased expression and mutation of ERCC1 gene may be the possible carcinogenic mechanism of CdCl2.

Animals ; Bronchi ; cytology ; Cadmium Chloride ; toxicity ; Cell Transformation, Neoplastic ; Cells, Cultured ; DNA-Binding Proteins ; genetics ; metabolism ; Endonucleases ; genetics ; metabolism ; Epithelial Cells ; cytology ; metabolism ; Exons ; Frameshift Mutation ; Humans ; Mice ; Mice, Nude ; RNA, Messenger ; metabolism

OBJECTIVETo explore the expression and sequence of human MutS homologue 2 (hMSH2) during different stages of human bronchial epithelial (16HBE) cells induced by cadmium chloride (CdCl2).

METHODSReverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical staining (SP method) were used to measure the hMSH2 mRNA and protein expression in 16HBE cells and its different passage cells treated by CdCl2 (the 5th, 15th, 35th passage, and neoplasm cells from nude mice's tumor tissue). hMSH2 exon 6, hMSH2 exon 7, hMSH2 exon 8, hMSH2 exon 9, hMSH2 exon 12 of the 16HBE cells and neoplasm cells from nude mice's tumor tissue were amplified by polymerase chain reactions (PCR). The amplified DNA strips were purified. Then the exons were detected by DNA analysis.

RESULTSDuring the passages of 16HBE cells treated with CdCl2, the expression of hMSH2 gene were decreased gradually. The hMSH2 gene mRNA and protein expression levels of the CdCl2 transformed 35th 16HBE cells and tumorigenic cells of nude mice significant decreased compared with non-transformed 16HBE cells (P < 0.01). In the tumorigenic cells of nude mice induced by CdCl2, there were thymine (T) deletion in 1st, 2nd and 7th site of hMSH2 exon 8, there were adenine (A) deletion in 20th and 182th site of hMSH2 exon 9, there were adenine (A) insertion in 241st site of hMSH2 exon 12. All the mutations were frame shift mutation.

CONCLUSIONThe expression decreased and the mutation of hMSH2 gene may be the possible carcinogenic mechanism for CdCl2.

Animals ; Bronchi ; cytology ; Cadmium Chloride ; toxicity ; Cell Line ; Cell Transformation, Neoplastic ; chemically induced ; genetics ; metabolism ; Epithelial Cells ; drug effects ; metabolism ; pathology ; Humans ; Mice ; Mice, Nude ; MutS Homolog 2 Protein ; genetics ; metabolism ; Mutation ; RNA, Messenger ; genetics

OBJECTIVETo develop an simple and sensitive method for detecting anti-coronavirus IgG antibodies in bat sera based on enzyme-linked immunosorbent assay (ELISA).

METHODSA commercial ELISA kit for detecting SARS-CoV antibody was modified for detecting coronavirus antibodies in bat serum samples. The second antibody in the kit was replaced with horseradish peroxidase-conjugated protein-A (HRP-SPA) based on the characteristics of binding between Staphylococcus aureus protein A (SPA) and mammal IgG Fc fragment. The sera of 55 fulvous fruit bats (Rousettus dasymallus) were tested using the SPA-ELISA.

RESULTSThe test results of the positive and negative controls in the kit and the serum samples from convalescent ;patient were consistent with expectation. Coronavirus antibody was detected in 2 out of the 55 bat serum samples. Serum neutralization test confirmed the validity of the SPA-ELISA method.

CONCLUSIONThis SPA-ELISA method is applicable for detecting coronavirus antibody in bat sera.

Animals ; Antibodies, Viral ; blood ; Chiroptera ; virology ; Coronavirus ; immunology ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; methods ; Immunoglobulin G ; blood

Various stem cells, including neural stem cells (NSCs), have been extensively studied in stroke models, but how to increase neuronal differentiation rate of NSCs remains unresolved, particularly in a damaged environment. The purpose of this study was to investigate the effects of cerebral microvascular endothelial cells (CMECs) on the neurogenesis of NSCs with or without oxygen-glucose deprivation (OGD). The NSCs acquired from primary culture were immunostained to prove cell purity. Survival and proliferation of NSCs were determined after the co-culture with CMECs for 7 days. After removing the CMECs, NSCs were randomly divided into two groups as follows: OGD and non-OGD groups. Both groups were maintained in differentiation culture for 4 days to evaluate the differentiation rate. Mouse embryo fibroblast (MEF) cells co-cultured with NSCs served as control group. NSCs co-cultured with CMECs had an increase in size (on the 7th day: 89.80±26.12 μm vs. 73.08±15.01 μm, P<0.001) (n=12) and number [on the 7th day: 6.33±5.61/high power objective (HP) vs. 2.23±1.61/HP, P<0.001] (n=12) as compared with those co-cultured with MEF cells. After further differentiation culture for 4 days, NSCs co-cultured with CMECs had an increase in neuronal differentiation rate in OGD and non-OGD groups, but not in the control group (15.16% and 16.07% vs. 8.81%; both P<0.001) (n=6). This study provided evidence that OGD could not alter the effects of CMECs in promoting the neuronal differentiation potential of NSCs. These findings may have important implications for the development of new cell therapies for cerebral vascular diseases.
Animals ; Animals, Newborn ; Brain ; blood supply ; Cell Differentiation ; physiology ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; methods ; Endothelial Cells ; cytology ; metabolism ; Glucose ; metabolism ; Mice ; Mice, Inbred C57BL ; Microvessels ; cytology ; metabolism ; Neural Stem Cells ; cytology ; metabolism ; Oxygen ; metabolism