10.3969/j.issn.1000-8179.2013.12.014

Objective: To investigate the safety of transgenic human lung adenocarcinoma cell line SPC-A-1/IL-2 as tumor vaccine. Methods: IL-2 gene was introduced into human lung adenocarcinoma cell line SPC-A-1 and was expressed stably on the basis of the construction of retroviral packing cell line PA317/pLIL-2SN.The mutagenesis of both the DNA and supernatant of SPC-A-1/IL-2 in the and in vitro was tested by means of genetic toxicological techniques.Results:The result indicated that mutagenesis of both the DNA and the supernatant of transgenic cell SPC-A-1/IL-2 was not observed. Conclusion: The initial experiment suggested that the application of transgenic cell SPC-A-l/IL-2 as tumor vaccine was bisically safe and reliable.

Objective To investigate the value of diffusion-weighted magnetic resonance imaging in the differential diagnosis of primary gallbladder cancer with liver invasion and primary hepatocellular carcinoma (HCC) with gallbladder invasion. Methods From January 2009 to October 2010, 11 patients with primary gallbladder cancer and 19 patients with primary HCC were admitted to the PLA General Hospital. The clinical data of the 30 patients were retrospectively analyzed. All patients underwent diffusion-weighted magnetic resonance imaging with b value of 800 s/mm2, and the receiver operating curve (ROC) was drawn. The apparent diffusion coefficient (ADC) values of the patients with gallbladder cancer and HCC were compared by independent sample t test. Results Thirty tumors were detected in the 30 patients. All tumors showed high signal on DWI, slightly low signal on T1 WI and slightly high signal on T2 WI. The foci of 11 patients with primary gallbladder cancer were at the gallbladder fossa, and 10 of them had liver involvement. The mean ADC value of the 11 patients was (0.89 ±0. 14)mm2/s. Of the 19 patients with primary HCC, the foci of 15 patients were at the right lobe of liver, and 4were at the left lobe. The mean ADC value of the 19 patients was (1.04 ±0.18)mm2/s. There was a significant difference in the ADC value between patients with primary gallbladder cancer and those with primary HCC ( t =2.425, P＜0. 05). The area under the ROC was 0. 756 (95% confidence interval: 0.577-0. 935), and the sensitivity and specificity were 68.4% and 81.8%, respectively, when the threshold value was 0.96 mm2/s.Conclusion The ADC value of patients with primary gallbladder cancer is lower than those with primary HCC when the b value is 800 s/mm2, which is helpful in the differential diagnosis of primary gallbladder cancer and primary HCC.

Objective To assess the diagnostic value of MRI on multiple focal nodular hyperplasia (FNH) of the liver. Methods MR images of 9 cases with pathological-confirmed multiple FNH were retrospectively analyzed. MRI features of the lesions were correlated with pathological findings. Results Multiple FNH was considered in all these 9 cases. Among them, the primary diagnosis was FNH in 5,hepatic adenoma in 3 and fibrolamellar hepatocellular carcinoma in 1 case. A total of 31 lesions were detected in the 9 cases. On T2WI, 19 lesions presented slightly high-signal intensity, and the other 12 presented iso-signal intensity. On T1WI, 12 lesions presented slightly low-signal intensity, 7 presented iso-signal intensity, and the other 12 presented high-signal intensity. On opposed-phase, the signal intensity of 1 lesion dropped unevenly. After bolus injection of contrast agent Gd-DTPA, in hepatic arterial phase 18 lesions showed mild to marked heterogeneous enhancement, 11 showed marked homogeneous enhancement, 1 showed moderate ring-like enhancement, and the last one did not have obvious enhancement In portal venous and delayed phase, all the lesions turned to iso- or slightly high-signal intensity gradually. Sixteen of 31 lesions presented central scar, which demonstrated mild star-like enhancement in delayed phase. Conclusion Multiple FNH presented certain MRI features, which contributed to the preoperative diagnosis.

OBJECTIVETo explore the changes of anticancer efficiency of 5'-deoxy-5-fluorouridine (5'-DFUR) and 5-fluorouracil (5-Fu) in colorectal cancer cell line HT29 and LS174T cells after transfection of thymidine phosphorylase (TP) cDNA with a lentiviral vector.

METHODSTP cDNA was transfected into human colorectal cancer cell lines HT29 and LS174T with the lentiviral vector pLenti6.3-MCS-IRES2-EGFP, and the transfection efficiency of the two cell lines passed 5 generations was analyzed by flow cytometry. The expression of TP protein and the relative quantitative expression of TP mRNA in these 2 cell lines were detected by Western blot and RT-PCR, respectively. The 50% inhibitory concentration (IC50) of 5'-DFUR and 5-Fu in both HT29 and LS174T parent cells and TP-transfected cells were assessed by MTS assay. Finally, the concentration of converted 5-Fu was detected by high performance liquid chromatography (HPLC) either in the medium containing a series of concentrations of 5'-DFUR, in which HT29/HT29-TP or LS174T/LS174T-TP cells were cultured, or in the cell culture lysates.

RESULTSThe HT29 and LS174T cells transfected with human TP cDNA were monitored for 5 generations, and their transfection efficiency was about 95.0%. Immunohistochemical staining showed that both the parent cells and TP-transfected HT29 and LS174T cells were TP-positive, while vector-transfected cells were TP-negative. Western blotting showed that the TP protein expression in HT29-TP and LS174T-TP cells were significantly increased compared with that in their parents cells. The relative quantity (RQ) values of TP mRNA in HT29-TP and LS174T-TP cells were 8.45 ± 0.15 and 2 615.02 ± 253.97, respectively, which were significantly higher than that in their parents cells (P < 0.01). The IC50 values of 5'-DFUR on HT29-TP cells and its parents cell were (14.33 ± 0.74) µmol/L and (707.66 ± 5.66) µmol/L, respectively (P < 0.05), while (0.59 ± 0.11) µmol/L in LS174T-TP cells and (239.20 ± 21.83) µmol/L in its parent cells, respectively (P < 0.05). The IC50 values of 5-Fu of HT29-TP cells and its parents cells were (5.42 ± 0.75) µmol/L and (14.19 ± 0.97) µmol/L, respectively (P < 0.05), while (4.41 ± 0.96)µmol/L in LS174T-TP cells and (16.42 ± 2.12)µmol/L in its parents cells, respectively (P < 0.05). The HPLC results showed that the 5-Fu concentration detected from media contained a series of concentrations of 5'-DFUR for culturing HT29-TP and LS174T-TP cells were 12.2 to 28.7-folds and 13.1 to 23.6-folds, respectively, higher than that in their parents cells, (P < 0.01 for all). Otherwise, just a little of 5-Fu was detected in the two TP-transfected cells lysate, about 0.9% to 4.2% of 5-Fu detected in the media of the same cultured cells, whereas no 5-Fu was detected in the two parent cell lysates.

CONCLUSIONSTransfection of TP cDNA into colorectal cancer cell lines HT29 and LS174T with lentiviral vector can improve the expression of both TP mRNA and TP protein levels in passaged cells, enhance the conversion of 5-Fu from 5'-DFUR in the medium, and result in an enhanced anticancer effect on these two cell lines.

Antineoplastic Agents ; pharmacology ; Cell Line ; Cell Line, Tumor ; Colorectal Neoplasms ; genetics ; metabolism ; DNA, Complementary ; Floxuridine ; pharmacology ; Fluorouracil ; Genetic Vectors ; HT29 Cells ; Humans ; RNA, Messenger ; Thymidine Phosphorylase ; genetics ; metabolism ; Transfection

Objective To investigate the effect of fluoride exposure on expression of miRNA (miR)-200c and its target in human osteoblast Saos-2 cells.Methods Saos-2 cells were cultured in DMEM/F-12 medium and treated with fluoride (sodium fluoride,NaF).There were two groups including:control group (0 mg/L) and fluoride group (4 mg/L).Cells were harvested after 48 hours of culture with fluoride.The expression of miR-200c,the mRNA of alkaline phosphatase (ALP),osteocalcin (BGP),the target phosphatase and tensin homolog deleted on chromosome ten (PTEN) and dual-specific phosphatase 1 (DUSP1) of miR-200c was detected by qRT-PCR.The protein expression of PTEN and DUSP1 was detected by Western blotting.Results The expressions of ALP,BGP mRNA and miR-200c in Saos-2 cells in the fluoride group (23.60 ± 1.87,9.41 ± 0.94,8.61 ± 0.26) were higher than those in the control group (1.00 ± 0.11,1.00 ± 0.07,1.00 ± 0.12).The differences were statistically significant (t =-24.084,-18.388,-8.687,P ＜ 0.05).The mRNA expressions of PTEN and DUSP1 in the fluoride group (0.63 ± 0.02,0.38 ± 0.02) were lower than those in the control group (1.02 ± 0.24,1.02 ± 0.24).The differences were statistically significant (t =3.327,5.454,P ＜ 0.05).The protein expressions of PTEN and DUSP1 in Saos-2 cells in the fluoride group (1.19 ± 0.10,0.83 ± 0.07) were lower than those in the control group (1.81 ± 0.14,1.44 ± 0.25).The differences were statistically significant (t =6.250,4.171,P ＜ 0.05).Conclusion Exposure to fluorine may increase the expression of miR-200c in Saos-2 cells,and fluorine may act on PTEN and DUSP1 through miR-200c,downregulates the mRNA and protein expression levels of PTEN and DUSP1.

Objective:To evaluate the intelligence, movement and physical development of 2-month-old infants, and to explore the influencing factors of early infant development.Methods:A total of 203 infants aged 42 days who underwent physical examination in the Department of Neonatology of the Fourth Affiliated Hospital of Harbin Medical University from May to December 2021 were selected as subjects. At the age of 2 months, 184 infants were followed up for their intelligence, movement and physical development. The intelligence and movement development of infants were evaluated with the intelligence development scale. Their body length, weight, and head circumference were measured, and physical development was assessed by Z-score method. Logistic regression was used to analyze the influencing factors of intelligence, movement and physical development of 2-month-old infants.Results:The median mental developmental index (MDI) of 184 2-month-old infants was 101; the median psychomotor developmental index (PDI) was 89; the body length was (59.3 ± 2.0) cm, the body weight was (5.8 ± 0.6) kg, and the head circumference was (38.9 ± 1.1) cm. Multiple logistic regression analysis showed that the influencing factors of MDI were maternal delivery age and infant gestational age [odds ratio ( OR) = 0.849, 0.463, P < 0.05]; the influencing factors of PDI were maternal prenatal education and average sleep time per night during pregnancy ( OR = 0.512, 0.666, P < 0.05); the influencing factor of body mass index (BMI) was maternal iodine supplements during pregnancy ( OR = 2.858, P = 0.018); the influencing factor of length-for-age Z-score (LAZ) was maternal prenatal education during pregnancy ( OR = 0.265, P = 0.026); the influencing factors of weight-for-age Z-score (WAZ) included maternal average sleep time per night, the frequency of sleeping time exceeding 12 am and the average weekly exercise duration during pregnancy ( OR = 0.277, 1.106, 0.990, P < 0.05). Conclusion:Maternal delivery age, prenatal education, sleep duration, intake of iodine supplements, exercise duration during pregnancy, and infant gestational age are factors affecting the intelligence, movement and physical development of 2-month-old infants, which should be paid attention to and measures should be taken to improve the population quality.

Objective To study the effect of T-2 toxin on proliferation and cell cycle of rat chondrocytes,in order to provide a new idea in molecular mechanism of T-2 toxin-induced chondrocyte damage.Methods Primary chondrocytes of neonatal Wistar rats were isolated and stained by toluidine blue staining and type Ⅱ collagen immunofluorescence staining.The effects of different concentrations of T-2 toxin [0 (control),1,5,10,20,50,100 μg/L)] on proliferation of chondrocytes for 24 h were detected by cell counting kit-8 (CCK-8) method,and control,1 (low dose),5 (medium dose),and 10 μg/L (high dose) T-2 toxin were selected for subsequent experiment;cell cycle changes were detected by flow cytometry;Real-time PCR and Western blotting were used to detect the effects of T-2 toxin on mRNA and protein expressions of proliferating cell nuclear antigen (PCNA) and Cyclin D1 in chondrocytes.Results With increase of T-2 toxin concentration (control,1,5,10,20,50,100 μg/L),the cell survival rates [(100.00 ± 0.00)％,(93.12 ± 1.66)％,(77.12 ± 1.11)％,(59.44 ± 4.09)％,(46.64 ± 3.86)％,(38.15 ± 3.37)％,(33.79 ± 0.99)％] were decreased,and the differences were statistically significant (F =139.21,P ＜0.05).The percentages of quiescent phase/pre-DNA synthesis phase (G0/G1 phase) ceils in 1,5,10 μg/L T-2 toxin groups [(22.03 ± 0.42)％,(30.54 ± 2.61)％,(36.01 ± 1.51)％] were significantly higher than that in control group [(13.79 ± 1.65)％,P ＜ 0.05];the percentages of DNA synthesis phase (S phase) cells [(60.27 ± 3.53)％,(53.88 ±4.38)％,(49.55 ± 2.49)％] were significantly lower than that in control group [(76.72 ± 4.24)％,P ＜ 0.05].The differences of mRNA levels of PCNA and Cyclin D1 between groups were statistically significant (F =46.80,17.97,P ＜ 0.05),and 5,10 μg/L T-2 toxin groups (0.77 ± 0.13,0.79 ± 0.08,0.60 ± 0.07,0.56 ± 0.05) were lower than the control group (0.99 ± 0.02,1.01 ± 0.01,P ＜ 0.05).The expressions of PCNA protein in 5,10 μg/L T-2 toxin groups (0.69 ± 0.03,0.49 ± 0.03) were lower than that in control group (0.92 ± 0.05,P ＜ 0.05);the expressions of Cyclin D1 protein in 1,5,10 μg/L T-2 toxin groups (0.80 ± 0.06,0.60 ± 0.07,0.33 ± 0.13) were lower than that in control group (0.95 ± 0.07,P ＜ 0.05).Conclusion T-2 toxin can inhibit the proliferation of chondrocytes,which may be worked through influencing the expression of cell cycle protein,causing cell cycle arrest,thereby inhibiting DNA synthesis.