【 Abstract 】 Objective To observe the effect of disruption of hypoxia inducible factor-1 α (HIF-1 α ) pathway by small hairpin RNA (shRNA) on chemosensitivity of human hepatocellular carcinoma (HCC) cells and to reveal the correlative mechanisms. Methods Plasmid of pshRNA-HIF-1α was transfected into HepG2 cells by lipofectamine. HepG2/pshRNA-HIF-1α (HepG2/pshRNA) cell lines were obtained by selection of HepG2 cells in G418. Meanwhile, plasmid of empty vector (pHK) was transfected as a control (HepG2/pHK). The mRNA and protein expression levels of HIF-1α and mdr1 were investigated by RT-PCR and Western blot respectively. Using CoCl2 to simulate the hypoxia condition, growth inhibition and apoptosis rates of HepG2 cells under different dosages of chemotherapeutic agents (adriamycin) were measured by MTT assay and flow cytometry (FCM) ． ResultsCompared with HepG2/pHK cells, the mRNA and protein expression levels of HIF-1αand mdr1 were obviously down-regulated in HepG2/pshRNA cells. At the same time, the proliferation inhibition and apoptosis rates were evidently increased after transfection with pshRNA-HIF-1α(P＜0.05)，which decreased the expression of HIF-1αto 82.18% at mRNA level and 75.51% at protein level. There was no significant effect of transfection pHK (Pgt;0.05). Conclusion These data demonstrates that HIF-1α interference by shRNA increased the sensitivity of HCC chemotherapy and the reversal of multidrug resistance, which may be done by down-regulating the transcription of mdr1 and the translation of P-gp. Blocking HIF-1αin HCC cells may offer an new avenue for gene therapy．
Objective To investigate the effects of X-ray dose on the expressions of microRNA-221 （miR-221） and phosphatase and a tensin homolog deleted from chromosome10 （PTEN） in human colorectal carcinoma （CRC） cells. Methods Human CRC-derived cell line， Caco2， was cultured conventionally. The cells were divided into five groups and exposed to different doses of X-ray （0， 2， 4， 6， and 8 Gy） respectively. The total RNA and protein of the Caco2 cells were extracted after irradiation， and the miR-221 and PTEN mRNA expressions were detected by real-time RT-PCR.Moreover， the protein alteration of PTEN in Caco2 cells was detected by Western-blot analysis. Results The radiation dose of X-ray significantly affected the expressions of miR-221 and PTEN protein in human Caco2 cells in a dose-depen-dent manner. Moreover， the miR-221 expression level was up-regulated gradually with the increase of irradiation dose， on the contrary， the PTEN protein expression level was down-regulated gradually （P＜0.01）. Conclusion The radiation dose can affect the miR-221 and PTEN protein expression pattern in CRC cells.
ObjectiveTo investigate the clinical settings, antibiotic susceptibilities, management and outcomes of streptococcal endophthalmitis. MethodsA retrospective observational case series study. Fifty six eyes of 56 patients diagnosed with streptococcal endophthalmitis in Eye & ENT Hospital, Fudan University from 2012 to 2022 were collected. The treatment followed the general principles of relevant guidelines, including pars plana vitrectomy (PPV), vitreous injection of antibiotics (IVI), vitreous injection of glucocorticoids and systemic application of antibiotics. The follow-up time was (11.9±17.0) months. Patients' clinical characteristics, pathogenic distribution and antibiotic susceptibilities, treatment and outcomes in their medical records were retrospectively collected and analyzed. ResultsAll 56 patients had monocular onset, including 39 (69.6%, 39/56) males and 17 (30.4%, 17/56) females, 26 (46.4%, 26/56) with left eyes involved and 30 (53.6%, 30/56) with right eyes involved. Their average age was (25.0±24.4) years. Ocular trauma was the leading cause of streptococcal endophthalmitis (73.2%, 41/56), followed by ophthalmic surgery (23.2%, 13/56) and endogenous infection (3.6%, 2/56). The streptococcal species included Streptococcus viridans (50.0%, 28/56), Streptococcus pneumoniae (18/56, 32.1%) and β-hemolytic Streptococcus (17.9%, 10/56). The susceptibility rates of Streptococcus to penicillin, cefatriaxone, vancomycin and levofloxacin were 66.0%, 57.1%, 94.1% and 92.4%, respectively. Patients received PPV+IVI and IVI as initial treatment were 49 eyes (87.5%, 49/56) and 7 eyes (12.5%, 7/56), respectively. Vitreous injection of glucocorticoids were performed in 17 eyes (30.4%, 17/56); and systemic antibiotics were applied in 52 cases (92.9%, 52/56). At the final follow-up, 47 eyes were recorded with visual acuity. Twenty (35.7%, 20/56) had best corrected visual acuity (BCVA)≥0.05 and 27 (48.2%, 27/56) had BCVA ＜0.05, of which 1 (1.8%, 1/56) had an eyeball enucleation. The etiology of endophthalmitis, streptococcal species, initial treatment with PPV, vitreous injection of glucocorticoids, and systemic antibiotics did not significantly affect patients' visual outcomes (P＞0.05). Timely visit to the hospital after the onset of symptoms (≤3 days) was significantly associated with achieving a final BCVA above 0.05 (P=0.025). ConclusionsOcular trauma was the primary cause of streptococcal endophthalmitis. Streptococcus viridans is the most common pathogenic bacterium. Streptococci had high susceptibility to vancomycin, but patients' visual outcomes were poor.
Objective To explore the safety of neoadjuvant chemoradiotherapy combined with sphincter-preserving operation in treatment of locally advanced low rectal cancer. Methods The clinical data of thirty-four patients admitted into our hospital between June 2007 and June 2009 with T3 and T4 low rectal cancer treated by neoadjuvant chemoradiotherapy and sphincter-preserving operation were collected and analyzed retrospectively. Routine fraction of radiation was given with total dose of 40 Gy, five times a week, 2 Gy per fraction. Patients received oxaliplatin （150 mg/d1）, plus folinic (100 mg/d1-3) and 5FU (750 mg/d1-3) for total 1 cycles started from the 4th week of irradiation. Operation was performed 4 weeks after neoadjuvant therapy. Results After neoadjuvant therapy, all patients underwent surgical resection with average tumor size decreased by 41.2%, tumor T stage decreased in 67.6% （23/34) patients, and lymph nodenegative change rate was 58.8% (10/17). One patient had liver metastasis and one had local recurrence, but without stomal leak. And 88.2% (30/34) patients showed good function of sphincter. Conclusions Neoadjuvant chemoradiotherapy in advanced lower rectal cancer patients has shown its efficacy in down-staging, which is safe without increasing operation complications when combined with sphincterpreserving surgery.
Objective To explore effect of DLL4 gene in MCF-7 cells of human breast cancer which was inhibitted by short hair in RNA （shRNA） on inducing apoptosis and chemosensitivity to docetaxel. Methods Specific shRNA was designed in accordance with DLL4 gene and transfected into MCF-7 cells of human breast cancer with liposomal （Lip-shRNA group）， MCF-7 cells transfected with only liposomal as Lip group， and control group without any treatment. Expressions of DLL4 protein in 3 groups were detected by immunohistochemical method， apoptosis and cell cycle distribution were examined by flow cytometry （FCM）. Proliferations and sensitivity of MCF-7 cells to docetaxel in 3 groups were determined by methylthiazoyl-tetrazolium bromide （MTT）. Results The averages optical density value and rate of positive area of Lip-shRNA group were significantly lower than that of other 2 groups （P＜0.01）. The levels of A value at 24h， 48h， and 72h in Lip-shRNA group were significantly lower than that of other 2 groups （P＜0.01）. Rates of cell apoptosis at 24h， 48h， 72h and 96 h in Lip-shRNA group were significantly higher than that of other 2 goups （P＜0.05），and ratio of G2/M was higher too （P＜0.05）. IC50 of Lip-shRNA group to docetaxel was significantly lower than that of other 2 groups （P＜0.05）. Conclusions The RNA interference technology can effectively block the expression of DLL4 gene. Inhibition of DLL4/Notch signaling pathway can lead to proliferation inhibition of cancer cell and a concomitant increase in cells undergoing apoptosis， and can enhance the cell sensitivity to docetaxel. DLL4 may be an important target for therapeutic approach of breast cancer.
We have studied the radiosenstivity of retinoblastoma cell [inc: HXO Rb~4,and found that the ceil growth reduced markedly after being treated by 3GyT-ray. From both clone for mation method and MTT assay,we identify that HXO-Rb44 cell is radiosensitive to T-ray.Oxygen can increase the radiosensitivity of HXO-Rb44 cell, but decrease the repair of sublethal damage.Oxygen enchaneement ratio(OER)is 2.77~3.01. （Chin J Ocul Fundus Dis,1994,10:217-219）
Objective To evaluate the utility of collagen-gel droplet embedded-culture drug sensitivity test (CD-DST) in pancreatic carcinoma cell by compared with WST-8. Methods The chemosensitivity to 5-fluorouracil (5-FU), gemzar (GEM) and oxaliplatin (OXA) of pancreatic adenocarcinoma cells SW1990, PCT-3 and ASPC-1 were tested by WST-8 and CD-DST respectively. Results In a certain living cell number range (500-10 000), there was a linear correlation (r=0.991 1, P＜0.05) between the integral optical density in CD-DST and the cell number. The inhibition ratios of three kinds of cell growth tested by CD-DST were higher than those tested by WST-8 (P＜0.05). The results of drug chemosensitivity to 5-FU， GEM and OXA detected by two methods were uniform. Conclusion The CD-DST can be used to assay the drug chemosensitivity in vitro for pancreatic carcinoma.
Survival data were widely used in oncology clinical trials. The methods used, such as the log-rank test and Cox regression model, should meet the assumption of proportional hazards. However, the survival data with non-proportional hazard (NPH) are also quite usual, which will decrease the power of these methods and conceal the true treatment effect. Therefore, during the trial design, we need to test the proportional hazard assumption and plan different analysis methods for different testing results. This paper introduces some methods that are widely used for proportional hazard testing, and summarizes the application condition, advantages and disadvantages of analysis methods for non-proportional hazard survival data. When the non-proportional hazard occurs, we need to choose the suitable method case by case and to be cautious in the interpretation of the results.
Objective To investigate the radiation-sensitizing effects of Ku80 silencing by siRNA interference for A549 lung cancer cells. Methods The sequences of Ku80 siRNA and negative siRNA were chemically synthesized and transfected into A549 lung cancer cells by lipofectamine. RT-PCR and Western bolt analysis were used to determine Ku80 gene expression. The transfected cells in culture dishes were irradiated with X ray at doses of 2 Gy, 4 Gy, 6 Gy, 8 Gy, 10 Gy, respectively. Once all treatments were completed, the cells were processed with the colony formation assay. Results RT-PCR detection showed that Ku80 mRNA levels in A549 lung cancer cells were reduced after transfected with Ku80 siRNA at 24 h, 48 h and 72 h time points. Western blot analysis showed that Ku80 protein content decreased at 48 h and 72 h time points compared with the control group ( P lt; 0. 05 ) . Cloning formation assay indicated that radiosensitivity of A549 lung cancer cells was enhanced after transfected with Ku80 siRNA. Conclusion Ku80 siRNA can effectively inhibit Ku80 gene expression of A549 lung cancer cells, and therefore enhance its radiosensitivity.