[Cloning, expression, purification and identification of multi-potent transcription factor CTCF recombinant and its segments].

OBJECTIVE: To clone ctc f cDNA, N, Zn and C fragments separately into expresstion vector, purify and identify the expressed proteins. METHODS: Using the recombinant plasmid pGEM7Zf (-)-ctc f as template for PCR, pGEX-4T-2-ctc f, pGEX-4T-2-ctc f-N, pGEX-4T-2- ctc f-Zn and pGEX-4T-2- ctc f-C recombinants were constructed successfully. After transformed into E. coli BL21 cells, the recombinants were confirmed by enzyme digestion and sequence analysis. After optimizing the IPTG inducing condition, the purified GST fusion proteins with affinity chromatography were conformed by Far-Western blotting. RESULTS: The recombinant plasmids pGEX-4T-2-ctc f, pGEX-4T-2- ctc f-N, pGEX-4T-2-ctc f-Zn and pGEX-4T-2-ctc f-C were confirmed by restriction enzyme assay and sequencing. All GST fusion proteins, CTCF, CTCF-N, CTCF-Zn and CTCF-C were successfully expressed at the optimal parameters and purified with affinity chromatography, and specifically recognized by anti-GST antibody. CONCLUSION: Ctc f, ctc f-n, ctc f-Zn and ctc f-c gene recombinants were constructed successfully and their corresponding fusion proteins were successfully purified with affinity chromatography and identified.

[Study of mental disorder due to brain damage].

OBJECTIVE: To study the incidence rate, pattern and affective factors of mental disorder due to brain damage. METHODS: According to CCMD-2-R, 388 subjects with traumatic brain damage in the Psychiatric Hospital of Huainan city within last 5 years were assessed by three psychiatrists 6 months to 1 year after brain injury. RESULTS: (1) 74.2% of the mental disordered due to brain injury have intellectual impairment, most of them is mild; (2) The intracranial hematoma, brain stem injury, brain injury extent, GCS, complicated mental disorder, and education have great effect on intellectual impairment; (3) There is an intimate relationship between the intellectual impairment and the brain stem injury, intracranial hematoma, GCS, brain injury extent, and unconsciousness time. There is an intimate relationship between the mental symptom and the brain injury extent, contusion and laceration of brain, frontal lobe injury, and intracranial hematoma. There is also an intimate relationship between the personality change and the frontal lobe injury, unfolding brain case treatment, and intracranial hematoma. CONCLUSION: To assess overall mental disorder should rely on the characteristics of craniocerebral injuries.

Hypomethylated and hypermethylated profiles of H19DMR are associated with the aberrant imprinting of IGF2 and H19 in human hepatocellular carcinoma.

In this study, 39 human hepatocellular carcinoma (HCC) tissues and 7 normal adult liver tissues were screened for heterozygous polymorphisms in IGF2, H19, and the differentially methylated region of H19 (H19DMR) using PCR-RFLP and PCR sequencing. The imprinting of IGF2 and H19 was examined by RT-PCR-RFLP, while the methylation profile of H19DMR was detected by bisulfite sequencing from every informative sample. Of the informative HCC samples 47.06% (8 of 17) demonstrated a gain of imprinting of IGF2, and 21.74% (5 of 23) of the informative HCC samples demonstrated a loss of imprinting of H19. Interestingly, we found three methylation profiles for H19DMR in the informative HCC samples: hyper-, medium-, and hypomethylated profiles. Furthermore, the hypomethylated and hypermethylated profiles were immediately associated with aberrant imprinting of IGF2 and H19.

[The aberrant imprinting of insulin-like growth factor II and H19 in human hepatocellular carcinoma].

OBJECTIVE: To investigate the allelic expression of IGF2/H19 in human hepatocellular carcinoma and to analyze the relationship between the imprint status of IGF2 and that of H19. METHODS: Heterozygotes of IGF2 and of H19 were identified by restriction fragment length polymorphism (RFLP). Allelic expression was detected by reverse transcription-polymerase chain reaction (RT-PCR) and RFLP. RESULTS: Forty seven point one percent (47.1%, 8 of 17) of HCC samples were demonstrated to have the gain of imprinting (GOI) of in IGF2 gene, and 45.5% (5 of 11) of HCC samples were found to have the loss of imprinting (LOI) for H19 gene. No relationship was observed between the imprinting status of IGF2 and that of H19. CONCLUSIONS: IGF2 GOI and H19 LOI are common in HCC, and the imprinting of IGF2 could be independent from that of H19 in adult liver.

[Research on promoter region methylation status of tumor repressor gene P16 in human hepatocellular carcinoma by using methylation sensitive restriction enzyme and semi-nested PCR assay].

OBJECTIVE: To construct a combination method of methylation sensitive restriction enzyme and semi-nested touch down PCR assay for studying the promoter region methylation status of P16 gene in human hepatocellular carcinoma. METHODS: According to the sequence of CpG rich promoter region of P16 gene, three primers were designed and synthesized for semi-nested touch down PCR assay to examine the promoter region methylation status of P16 gene. 340 bp segment of this region was cloned into vector pMD18-T; the plasmid was transformed into E. coli JM109 to harvest an extended quantity, then the plasmid was treated by CpG methylase M. Sss I, the methylated plasmid was named P16Pm+. This P16Pm+ is validated by digestion of Hpa II and is employed in studying the specificity and sensitivity of this constructed method. After construction, the method was used to examine the promoter region methylation status in P16 gene of 40 DNA samples from human HCCs and three DNA samples from normal human liver tissue. RESULTS: It was confirmed that the specificity and sensitivity of this method are solid and reliable (100 fg). It was found that 12/40 (30%) of hepatocellular carcinoma showed promoter region methylation in P16 gene whereas none (0/3) of the normal tissues was methylated in the promoter region in P16 gene. CONCLUSION: Promoter region methylation in P16 gene may take part in human hepatocellular carcinogenesis. The constructed method is simple, cost-effective and is of high specificity and sensitivity, thus suggesting its potential application to detecting promoter methylation in population-based studies.

[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.

[P53 codon 72 polymorphism in cervical cancers and its correlation with HPV16,18E6].

OBJECTIVES: To examine p53 codon 72 polymorphism in cervical cancers and its correlation with HPV16/18 E6. METHODS: Cervical specimens were taken from 81 patients with cervical squamous cancer, 18 patients with cervical adenocarcinoma, 88 patients with CIN II - III and 60 patients without cancers. PCR was used to examine the p53 genotypes and the expression of HPV16 and 18 E6. RESULTS: The frequencies of p53 Arg homozygosity in cervical squamous cancer, cervical adenocarcinoma and CIN (II - III) were 58.020%, 55.55% and 59.09% respectively, greater than those of p53 Arg/Pro heterozygosity (30.86%, 27.78%, 21.59%) and those of p53 Pro homozygosity (11.12%, 16.67%, 19.32%). The normal cervical samples also showed less frequency of p53 Arg homozygosity (23.33%) than cervical squamous cancer. There were no significant differences in the frequencies of p53 Arg homozygosity, p53 Arg/Pro heterozygosity and p53 Pro homozygosity (23.33%, 40.00% and 36.67% respectively). The frequency of HPV16,18 E6-positive in cervical cancer and CIN was much higher than that in control group (81.84%, 50.00% and 53.41%) for the normal cervical samples. The expression of HPV16 and 18 E6 in cervical squamous cancers was more frequent than in CIN. The frequency of p53 Arg homozygosity in HRHPV E6-positive cervical squamous cancers (64.06%) was greater than in HRHPV E6-negative cervical squamous cancers (35.29%) and in HRHPV E6-positive normal samples (33.33%). The p53 codon 72 polymorphism showed no differences in samples with different FIGO staging and grades. CONCLUSION: p53 Arg homozygosity could serve as a risk indicator for the tumorigenesis of cervix. In combination with HRHPV E6, it might be able to predict the progression of cervical lesions. p53 codon 72 polymorphism is not associated with FIGO staging and grades of cervical cancers.

[Growth inhibition and demethylation by a component of natural drug, CDP, in cancer cell lines].

OBJECTIVE: To investigate the inhibition of tumor cell lines' growth and the demethylation of p16 gene by a component of natural drug, CDP. METHODS: Human liver cancer cell line Huh-7 and breast cancer cell line T47D were treated with the CDP and DNA methyltransferase inhibitor, 5-azacytidine (5-aza-C). The inhibition of growth was assayed by MTT; the methylation status of p16 gene promoter was analyzed by MSP. RESULTS: 1. The CpG islands in promoter of p16 gene were identified as completely methylated in Huh-7 and T47D. 2. CDP and 5-aza-C inhibited the proliferation of Huh-7 and T47D cell lines in a dose, time-dependent mode. 3. Methylation-specific bands of CpG islands in p16 gene' promoter were still existed in Huh-7 and T47D, while unmethylation-specific bands appeared in Huh-7 after the cells being treated with 25, 50, 75 and 100 micromol/ L of CDP for 6 days as well as in T47D after the cells being treated with 50, 75 and 100 micromol/L of CDP for 6 days. 4. After the cells being treated with 50 micromol/L of CDP, the methylation-specific bands of CpG island in p16 gene' promoter were still existed in Huh-7 and T47D; unmethylation-specific bands appeared at 12 h and lasted to 144 h in Huh-7 while at 72 h and lasted to 144 h in T47D. CONCLUSION: CDP has inhibition effect on the cancer cell lines and has the ability to reverse the hypermethylated p16 gene promoter. These results suggest that CDP has great potential in the development of effective anticancer drugs.

[Detection of aberrant promoter CpG islands hypermethylation of GSTP1 in human primary hepatocellular carcinomas].

OBJECTIVE: To detect the aberrant promoter CpG islands hypermethylation status of GSTP1 gene in hepatocellular carcinomas (HCC) and to assess its significant role in hepatocarcinogenesis. METHODS: Surgically resected cancerous and non-cancerous liver tissues of 26 hepatocellular carcinoma patients were obtained from West China Hospital, and 11 peripheral blood samples from healthy donors as negative control were collected. Breast cancer cell line MCF-7 with CpG islands hypermethylation of GSTP1 as positive control was obtained from the Cell Bank of Chinese Academy of Sciences in Shanghai. All genomic DNA were extracted using common phenolchloroform approach, and the 5' CpG islands methylation status of GSTP1 gene was studied by methylation-specific polymerase chain reaction (MSP). RESULTS: GSTP1 gene promoter CpG island hyperthylation was detected in 88.5% (23/26) of cancerous tissues and in 69% (18/26) of corresponding non-cancerous tissues from the 26 HCC patients. None of the 11 control samples were methylation positive. CONCLUSION: The data indicates that the detection of GSTP1 gene methyl-lation may be a valuable biomarker by MSP for HCC early diagnosis and disease monitoring.

[Research on the growth inhibition of T47D cell and reversion of p16 hypermethylation by CDP].

OBJECTIVE: To inquire into the mechanism of CDP (a purified component of Chinese crude drug) in inhibiting the breast cancer T47D cell growth. METHODS: T47D cells were treated with different concentration of 5-azacytidine (5-aza-CR) and CDP for several days. The growth rate was assessed by cell proliferation experiment (MTT colorimetric assay). The changes in apoptic peak and cell cycle distribution were detected by flow cytometry (FCM). The levels of methylation and unmethylation status of p16 were detected by methylation specific PCR (MSP). RESULTS: After treatment with the two drugs, the cell growth rate decreased in a dose and time dependent manner (P<0.05). The cell cycle was influenced by the well-chosen concentration of 5-aza-CR (2 micromol/L) and CDP (50 micromol/L): the cell number increased from 65.1% to 71.3%, 84.3% in G0/G1 phase and decreased from 19.4% to 14.3%, 7.2% in S phase. Demethylation on p16 gene occurred after treatment with any of the two drugs for 6 days. CONCLUSION: CDP can reverse p16 hypermethylation and may hence inhibit the proliferation of tumor cells.

[The imprinting status and expression of insulin-like growth factor 2 gene in human hepatocellular carcinoma].

OBJECTIVE: To study the imprinting status and expression level of insulin-like growth factor 2 (IGF2) gene in hepatocellular carcinoma and to provide a clue for elucidating the mechanism of carcinogenesis of hepatocellular carcinoma. METHODS: The heterozygote status of IGF2 gene was detected by restriction fragment length polymorphism. The imprinting status and expression level of IGF2 were evaluated by semi-quantitative reverse transcription-polymerase chain reaction. RESULTS: It was found that 10/18 (55.6%) of hepatocellular carcinoma showed the gain of imprinting (GOI), with 6/10 (60%) adjacent cirrhosis of liver tissues also displaying GOI of IGF2. Overexpression of IGF2 in cancer tissues was detected in 9/18 (50%) samples, but no significant difference was observed among each imprinting status. CONCLUSION: GOI of IGF2 may take part in human hepatocellular carcinogenesis.

[Impact of p16INK4A and p15INK4B on human hepatocellular carcinoma cell proliferation and apoptosis].

OBJECTIVE: Both tumor suppressor p16INK4A and p15INK4B are members of INK family of CDK inhibitor. Although the role of p16 has been well documented, the role of p15 and its signaling pathway remain less well studied. This study was aimed to assess the effect of p16 and p15 on hepatocarcinoma cell lines with different status of Rb gene. METHODS: After identification of the genetic status of p16, p15 as well as Rb of human hepatocellular carcinoma (HCC) cell lines BEL7402, SMMC7721 with the use of multiple PCR, the eukaryotic expression p16 and p15 recombinants pXJ-p16 and pXJ-p15 were constructed, respectively. The existence of exogenous p16, p15 genes, and the expression of p16 and p15 were assayed by means of PCR and RNA dot blotting. Finally, the proliferation and apoptosis were studied by using MTT, colony formation assay and flow cytometry. RESULTS: Neither deletion of p16 nor p15 was detected in the two cell lines. However, Rb exons 14-16 instead of exons 22-23 deletion existed in SMMC7721. The increased mRNA expression level of p16 was found in BEL7402-p16 and SMMC7721-p16, while increased mRNA expression level of p15 was found in BEL7402-p15. The endogenous p16 and p15 genes were transcripted at low level. The cell growth and colony formation were decreased in BEL7402-p15, compared with either mock cell BEL7402 or vector control cell BEL7402-pXJ. Also shown in this study were an altered G1 phase population from 37.7% to 43.6%, an S phase population from 22% to 13% (P<0.05), and a Sub G1 peak (apoptosis peak) in BEL7402-p15. Conversely, BEL7402-p16 with endogenous p16 gene showed neither difference in cell cycle population nor difference in colony formation rate, compared with control cell groups. Additionally, SMMC7721-p16 cell growth was not inhibited by exogenous p16 gene. CONCLUSION: p15 significantly arrested cell proliferation and induced apoptosis in BEL7402 in vitro, and the function was not influenced by endogenous p15 gene. The inhibition of cell growth by p16 on HCC cells could be dependent on intact RB pathway.

Association of low p16INK4a and p15INK4b mRNAs expression with their CpG islands methylation with human hepatocellular carcinogenesis.

AIM: To study the significance of p16 and p15 transcription suppression with hypermethylation of their genes' 5'CpG islands during human hepatocellular carcinogenesis. METHODS: The mRNA expression levels of p16 and p15 genes were evaluated in cancerous, para-cancerous and non-cancerous tissues of 20 HCC, 3 normal liver tissues from 3 accidentally died healthy adults using semi-quantitatively Northern blot. The methylation status was also assessed with methylation specific PCR. RESULTS: p16 mRNA expression level was decreased in the cancerous tissues in 60% (12/20) of HCC patients, of which 2 cases had no p16 mRNA detected, 5 cases (25%) displayed variation in the order of cancerous

[Value of diagnosing micrometastasis by nested RT-PCR in the peripheral blood and bone marrow in non-small cell lung cancer patients].

OBJECTIVE: To evaluate the specificity, sensitivity and their clinical significance of detecting CK19 mRNA expression by nested RT-PCR for molecular diagnosis of micrometastasis in the peripheral blood and bone marrow of patients with non-small cell lung cancer. METHODS: CK19 mRNA expression was detected by nested RT-PCR in peripheral blood and bone marrow samples from 59 lung cancer patients, with samples of 11 benign pulmonary lesion patients and 20 healthy adults as control. RESULTS: The sensitivity of nested RT-PCR was 10(-6). The positive rates of micrometastasis were 33.89% (20/59) in peripheral blood and 22.03% (13/59) in bone marrow, with a highly positive correlation existing between the two groups (P < 0.05). The micrometastasis in peripheral blood and bone marrow was closely correlated with the pathological classification and cell differentiation (P < 0.05) and P-TNM stage (P < 0.01). No CK19 mRNA expression was found in the samples from patients with benign pulmonary lesion or healthy adult volunteers. CONCLUSION: The peripheral blood and bone marrow from patients with non-small cell lung cancer possesses micrometastasis that can not be detected by common methods. Nested RT-PCR technique shows favorable specificity and sensitivity in detecting the condition with definite clinical prospects.

Equilibrium bed-concentration of nonuniform sediment.

Knowledge of the equilibrium bed-concentration is vital to mathematical modeling of the river-bed deformation associated with suspended load but previous investigations only dealt with the reference concentration of uniform sediment because of difficulties in observation of the bed-concentration. This work is a first attempt to develop a theoretical formula for the equilibrium bed-concentration of any fraction of nonuniform sediment defined at the bed-surface. The formula is based on a stochastic-mechanistic model for the exchange of nonuniform sediment near the bed, and described as a function of incipient motion probability, non-ceasing probability, pick-up probability, and the ratio of the average single-step continuous motion time to static time. Comparison of bed-concentration calculated from the proposed formula with the measured data showed satisfactory agreement, indicating the present formula can be used for solving the differential equation governing the motion of suspended load.