Objective To investigate the feasibility of using clustered regularly interspaced short palindromic repeats ( CRISPR)-mediated genome editing to downregulate the expression of programmed cell death protein 1 (PD-1) on primary T cells by using a lentivirus delivery system. Methods Lentivirus vec-tors pLentiCRISPR A1-A6 containing different PD-1 genomic DNA sequences as single guide RNA ( sgRNA) for Cas9 targeting were constructed individually. The lentivirus vectors were tranduced into primary CD4 T cells. Flow cytometry analysis was performed to detect the expression of PD-1 for evaluating the knockout ef-ficiency. Results The lentivirus vectors pLentiCRISPR A1-A6 carrying six different target sites were con-structed and respectively tranduced into primary CD4 T cells. The expression of PD-1 accompanied with the activation of T cells. Co-expression of CD25 and PD-1 was observed on activated T cells. All of the six sites could be targeted by Cas9, of which A2 and A6 sites were more efficient in knocking out the gene encoding PD-1 with a rate of 19% and 29%, respectively. Conclusion This study suggests that it is feasible to knock out the expression of PD-1 on primary T cells by using CRISPR.

To investigate the metabolic rate and metabolites of 9-dehydro-17-dehydro-andrographolide, which is the main active ingredient in Xiyanping injection, by using the in vitro rat liver microsome incubation system. 9-dehydro-17-dehydro-andrographolide was incubated together with liver microsome mixed with NADPH. Its metabolic rate was studied by determining its residual concentrations with the UHPLC-MS/MS method; Its metabolites were identified by the UPLC-TOF-MS(E) method. The results showed that 9-dehydro-17-dehydro-andrographolide was metabolized faster than rat liver microsomes mixed with coenzymes, with t½ and CL of (19.7 ± 0.5) min and (35.1 ± 0.8) mL x min(-1) x g(-1) (protein), respectively. Based on the high resolution mass spectrum data and information from literatures, altogether nine metabolites of 9-dehydro-17-dehydro-andrographolide were identified in the incubation system, particularly hydroxylated and dehydrogenized products. The results of identification would provide a basis for screening out more active andrographolide derivatives.
Animals ; Chromatography, High Pressure Liquid ; Diterpenes ; chemistry ; metabolism ; Drugs, Chinese Herbal ; chemistry ; metabolism ; Microsomes, Liver ; chemistry ; metabolism ; Molecular Structure ; Rats ; Tandem Mass Spectrometry

Objective To investigate the effect of gender and age on the apparent diffusion coefficient (ADC) of normal adult pancreas.Methods A total of 383 patients with normal pancreas (290 male,93 female,range from 21 to 78 years of age) were enrolled in this study.The subjects were divided into four groups based on different age (≤40 years,41-50 years,51-60 years and ＞60 years) with patient number of 56,108,139 and 80,respectively.Breath-hold single-shot echo-planar DWI (b value =0,500 s/mm2) was performed to determine ADCs on all patients.The average ADCs was calculated by four ADCs measured from the head to tail part of the pancreas in each patient.Patients with different age or gender were analyzed by independent-samples t test.Effect of gender on ADCs was analyzed using the Mann-Whitney U test.Relationship between ADCs and age was analyzed using Spearman rank-order correlation test,and Kruskal-Wallis H test was used to compare the ADCs among 4 age groups.Results The median pancreatic ADC values in female group(n =93) [1.60 × 10-3 mm2/s (1.47 × 10-3-1.77 × 10-3) mm2/s] was higher than that in male group (n =290) [1.57 × 10-3mm2/s(1.41 × 10-3-1.74 × 10-3)mm2/s].Mann-Whitney U test results showed the mean ADCs was similar between the two groups (Z =1.335,P =0.182).The age distribution was similar between the male [(52 ± 10) years of age] and female [(51 ± 11) years of age]groups (t =0.267,P=0.790).The age spectrum showed that there was no correlation between the average ADC values and age (r =0.016,P =0.752).The median ADC values of the four age groups were 1.58 ×10-3,1.54 × 10-3,1.59 × 10-3 and 1.57 × 10-3 mm2/s,respectively.Kruskal-Wallis H test showed no significant difference of mean ADCs among the age groups (x2 =2.15,P =0.542).Conclusion There is no correlation of ADCs between age and gender in normal adult pancreas.

OBJECTIVETo summarize the surgical outcome of patients with small cell esophageal carcinoma(SCEC).

METHODSClinical data of patients with esophageal carcinoma were retrospectively collected from March 2000 to March 2011 at the Thoracic Surgery Department of the Peking University Cancer Hospital. Data included tumor characteristics, staging, treatment, response, short-term outcome, and long-term survival.

RESULTSA total of 546 patients with esophageal carcinoma were identified, among whom there were 15 patients with SCEC(2.7%). Fourteen cases received multimodality treatment based on operation and one underwent operation alone. Four patients had preoperative chemotherapy and 10 had postoperative chemotherapy. Four patients had postoperative radiation. After excluding one case of postoperative death within 3 months, the median overall survival was 14.3 months(range, 4 to 99 months), significantly worse than those with non-SCEC(42.2 months, P<0.05).

CONCLUSIONSCEC is rare and the outcomes are poor. It should be considered as a systematic disease.

Adult ; Aged ; Carcinoma, Small Cell ; surgery ; therapy ; Combined Modality Therapy ; Esophageal Neoplasms ; surgery ; therapy ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Treatment Outcome

[ ABSTRACT] AIM: To investigate the protective effect of autophagy on oxidized low density lipoprotein ( ox-LDL)-induced macrophage apoptosis and the underlying molecular mechanisms .METHODS:The RAW264.7 macropha-ges were pretreated with 3 mmol/L 3-methyladenine (3-MA), 1 μmol/L rapamycin (Rap) or 4 mmol/L 4-phenylbutyric acid ( PBA) respectively for 1 h and then treated with ox-LDL (100 mg/L) for 12 h.The cell viability and apoptosis were determined by MTT assay and flow cytometry with Annexin V-FITC/PI staining, respectively.The activities of lactate de-hydrogenase ( LDH) in the medium and caspase-3 in the cells were determined by detection kits .The protein levels of bec-lin-1 (a molecular marker of autophagy ), glucose-regulated protein 78 (GRP78, an endoplasmic reticulum stress marker) and C/EBP homologous protein ( CHOP, a key-signaling component of endoplasmic reticulum stress-induced apoptosis ) were examined by Western blot .Microtubule-associated protein 1 light chain 3 (LC3, another molecular marker of autoph-agy) was observed under laser scanning confocal microscope .RESULTS: Treatment of the RAW264.7 macrophages with ox-LDL at 100 mg/L for 12 h resulted in significant decrease in cell viability , and dramatic elevation in LDH leakage , cell apoptosis and caspase-3 activity, which were promoted by 3-MA (an autophagy inhibitor) and inhibited by Rap (an autoph-agy inducer ) .ox-LDL induced autophagy in the macrophages as assessed by beclin-1 upregulation and frequent granulation of LC3, which were inhibited by 3-MA and promoted by Rap.Interestingly, 3-MA enhanced, while Rap blocked, the CHOP upregulation induced by ox-LDL.Moreover , PBA ( endoplasmic reticulum stress inhibitor ) significantly inhibited ox-LDL-induced GRP78 upregulation and autophagy as determined by the attenuation of beclin-1 upregulation and frequent granula-tion of LC3.CONCLUSION: Endoplasmic reticulum stress mediates ox-LDL-induced autophagy in macrophages , and moderates activation of autophagy may protect macrophages from ox-LDL-induced apoptosis by inhibiting CHOP expression .

Objective To explore the effects of amniotic lacrimal duct stenting on the prevention of dry eye in castrated rabbits.Methods Thirtysix healthy male rabbits were selected,the third eyelid were cut off and antiinfection treatment were given,which were randomly divided into 3 groups (12 cases in each group),the castrated male rabbits models were made.Among them,group A was negative control group,group B was dry eye model group,group C was group of lacrimal amniotic membrane group.At 2 weeks before implantation of amniotic lacrimal duct stent,2 weeks,4 weeks and 6 weeks after implantation,the fluorescent (FL) examination,Western blot,Schirmer I examination,immunofluorescence staining and corneal confocal microscopy were performed.Results The levels of tear secretion and FL in the three groups among different time points were significantly different (F=7.126,P =0.009;F =9.658,P =0.016),and there were significant differences among three groups (F =12.582,P =0.005;F =13.187,P =0.013).The tendency of tear secretion and FL in the three groups were also significantly changed (F =8.531,P =0.007;F =10.652,P =0.019).The epithelial basal cells at 6 weeks after implantation in three groups were 3811 ±414,3820 ± 314,2789 ± 353,and the density of inflammatory cells was 266 ±28,266 ± 29,67 ± 13,there were significant differences among three groups (F =13.442,P =0.012;F =9.231,P =0.021).The K1 6 staining in the duct epithelium were negative,and the expression of α-SMA in the lacrimal duct tissue of group A,B and C was not changed at all time points after implantation of amniotic lacrimal stent,and there was no significant difference (F =14.681,P =0.002).Conclusion The amniotic lacrimal stent implantation has certain effect on the prevention of dry eye in rabbit.

OBJECTIVETo investigate the clinical and pathological characteristics, treatment methods, and prognosis of synchronous primary cancers of the endometrium and ovary.

METHODSThe clinical data of 43 patients with synchronous primary cancers of the endometrium and ovary were retrospectively reviewed. The survival was calculated by Kaplan-Meier method and compared using the log-rank test.

RESULTSThe median age at diagnosis was 49 years (range, 28-73 years). The most common symptoms were abnormal vaginal bleeding (69.8%) and abdominal or pelvic pain (44.2%).Pelvic masses were found in 39.5% of the patients and enlarged corpus in 27.9% at physical examination, while pelvic masses were found in 67.4% of the 43 patients (29 cases) and thickening or abnormal endometrium in 23.3% (10 cases) during ultrasound examination. Of 25 patients examined by CT/MRI, pelvic masses were found in 13 cases and enlarged uterus in 11 cases. All 15 patients who underwent endometrial biopsies were proven to have endometrial carcinomas. Serum CA125 level was found to be elevated in 22 of the 34 examined cases (64.7%) with a median value of 500 U/ml (range, 39-3439 U/ml). FIGO stages of endometrial carcinomas: IA 18 cases, IB 20 cases, IC 2 cases, IIA 3 cases; Stages of ovarian carcinomas: IA 19 cases, IB 4 cases, IC 7 cases, II 4 cases, III C 9 cases. Twenty-four patients (55.8%) were in stage I both endometrial and ovarian carcinomas. Thirty-one patients underwent total hysterectomy plus bilateral salpingo-oophorectomy with omentectomy and appendectomy, meanwhile, 12 patients had pelvic lymph node dissection. Thirty-eight of the 43 patients (88.4%) had a pathologically proven endometrial adenocarcinoma. The predominant ovarian histology was endometrioid or mixed tumor with endometrioid components (30/43, 69.8%). Postoperatively, 26 patients (60.5%) received adjuvant chemotherapy alone, 12 had chemotherapy plus radiotherapy, only one patient had radiation alone and the remaining 4 cases received no adjuvant treatment. The 3- and 5-year survival rates of the group were 87.4% and 71.1%, respectively. The 3- and 5-year survival rates of patients with both endometrioid and ovarian carcinomas were higher than that of those with non-endometrioid or mixed subtypes (93.8%, 82.0% vs. 79.7%, 69.0%). The 3-year and 5-year survival rates of patients with early stage disease were better than those of the other patients (93.3%, 93.3% vs. 69.7%, 36.7%). Recurrence developed in 15 patients (34.9%). It was showed by univariate analysis that lower CA125 level, early FIGO stage, and adjuvant chemotherapy plus radiotherapy significantly and positively affect the 5-year survival rates, while only early FIGO stage and chemotherapy plus radiotherapy were revealed by multivariate analysis as independent prognostic factors.

CONCLUSIONSynchronous primary cancers of the endometrium and ovary are different from either primary endometrial carcinoma or ovarian cancer, while it can usually be detected in early stage and with a good prognosis. The impact of the CA125 level on prognosis needs to be further studied. Surgical treatment alone may be enough for early stage patients. Chemotherapy plus radiotherapy may be necessary for advanced stage patients.

Adult ; Aged ; Carcinoma, Endometrioid ; blood ; pathology ; surgery ; therapy ; Chemotherapy, Adjuvant ; Endometrial Neoplasms ; blood ; pathology ; surgery ; therapy ; Female ; Humans ; Hysterectomy ; methods ; Lymph Node Excision ; Lymphatic Metastasis ; Middle Aged ; Neoplasm Recurrence, Local ; Neoplasm Staging ; Neoplasms, Multiple Primary ; blood ; pathology ; surgery ; therapy ; Ovarian Neoplasms ; blood ; pathology ; surgery ; therapy ; Proportional Hazards Models ; Proteins ; metabolism ; Radiotherapy, Adjuvant ; Retrospective Studies ; Survival Rate

OBJECTIVETo review the experience in the treatment of low-grade malignant endometrial stromal sarcoma.

METHODSThe data of 41 patients with low-grade malignant endometrial stromal sarcoma surgically treated between 1982 and 2004 were reviewed. Statistical analysis was carried out using chi(2) and Kaplan-Meier life table.

RESULTSOf these 41 patients, 24 suffered from irregular vaginal bleeding, and 30 had been diagnosed to have leiomyoma before treatment. Thirty patients but 11 underwent surgical management with uterus removed. Thirty-three patients received postoperative adjuvant therapy including radiation and/or chemotherapy. The 5-year and 10-year actuarial survival was 87. 5% and 77. 8%, respectively. Eighteen patients (43. 9%) developed recurrent disease, most of which in the pelvis. The mean time to recurrence was 31 months (range 6 to 78 months) with the median time of 26 months. The recurrent rate was 66.7% for patients whose ovarian function was reserved versus 37. 5% for those without reservation. Patients who received adjuvant therapy had a lower recurrent rate (30. 3%) than those who did not (87. 5%). The recurrent rate of the patients treated with postoperative adjuvant radiation was 32. 3% (10/31) versus 80% (8/10) for those patients without. The 5-year actuarial survival rate of patients with recurrent disease was 71. 8%.

CONCLUSIONLow-grade malignant endometrial stromal sarcoma has a good prognoses though dwarfed by higher late recurrence after initial treatment. Postoperative adjuvant radiation is helpful to reduce local recurrence. Endometrial stromal sarcoma;

Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Brachytherapy ; methods ; Cesium Radioisotopes ; therapeutic use ; Chemotherapy, Adjuvant ; methods ; Combined Modality Therapy ; Endometrial Neoplasms ; pathology ; therapy ; Female ; Follow-Up Studies ; Humans ; Hysterectomy ; methods ; Kaplan-Meier Estimate ; Lung Neoplasms ; secondary ; therapy ; Middle Aged ; Neoplasm Recurrence, Local ; Prognosis ; Radiotherapy, Adjuvant ; methods ; Retrospective Studies ; Sarcoma, Endometrial Stromal ; secondary ; therapy

OBJECTIVETo investigate the clinical characteristics of clear cell carcinoma of the ovary and to compare the survival of the patients treated by three different chemotherapy regimens.

METHODSBetween 1984 and 2005, the clinical data of 88 surgically treated patients with clear cell carcinoma of the ovary were retrospectively analyzed. Of the 88 patients, 55 (62.5%) had tumor in stage I, 2 in stage II, 22 in stage II, 3 in stage IV and 6 in indefinite stage. These patients underwent either bilateral salpingo-oophorectomy with hysterectomy and omemtectomy or cytoreduction surgery. Of 55 stage I patients, 20 received pelvic lymohadenectomy. All patients were given postoperative chemotherapy, 43 patients received CAP/CP, 33 paclitaxel combination with carboplatinum/cisplatin (TC/TP) and 12 CPT-11 plus MMC.

RESULTSThe response rate, recurrence rate, 3- and 5-year survival was 35.0%, 30.2% (13/43), 67.4% (29/43), 43.9% and 29.3%, respectively in patients treated with CAP/CP; 73.9%, 18.2% (6/33), 45.5% (15/33), 57.3% and 40.5%, respectively in the patients with TC/TP; 71.4%, 16.7% (2/12), 25.0% (3/12), 70.7% ( 3-yr survival, no available 5-yr survival), respectively in the patients with CPT-11 + MMC (P < 0.05). During follow-up, 47 (53.4%) patients were found to have recurrence, it was 45.4% (25/55) in stage I patients including 29.6% (8/27) in stage I a + I b and 60.7% (17/28) in stage I c, 75.0% (18/24) in stage II + III and 4/6 in the indefinite FIGO stage. The recurrences rate was 27.8% (5/18) in stage I patients with pelvic lymphadenectomy vs. 51.3% (19/37) in those without. It was 67.3% in 46 patients with elevated CA125, and 38.1% in the other 42 patients with normal or unavailable CA125 (P < 0.05). The overall 3- and 5-year survival rate of 88 patients was 48.7% and 40.9% , respectively, with 72.5% and 66.8% in stage I, 100.0% and 70.5% in stage Ia + Ib, 68.5% and 60.3% in stage Ic, 41.8% and 20.8% in stage II + III, 0 in stage IV (P < 0.05). The 3- and 5-year survival in stage I with pelvic lymphadenectomy was 88.5% and 75.8% vs. 70.3% and 65.1% in those without (P < 0.05). The 3- and 5-year survival of the patients with optimal (residual disease less than 2 cm) was 36.7% and 23.1% vs. 22.2% and 0 in those with suboptimal cytoreduction (P < 0.05), it was 46.8% and 38.8% in the patients with elevated CA125 vs. 46.7% and 43.5% in those with normal one (P > 0.05).

CONCLUSIONOur data show that ovarian clear cell cancer patient have a poor response to CAP/CP and may have a better response to TC/TP, especially to CPT-11 plus MMC. However, the overall prognosis is still poor and further clinical investigations are needed to improve it.

Adenocarcinoma, Clear Cell ; blood ; drug therapy ; pathology ; surgery ; Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; CA-125 Antigen ; blood ; Female ; Follow-Up Studies ; Humans ; Hysterectomy ; methods ; Lymph Node Excision ; Lymphatic Metastasis ; Middle Aged ; Neoplasm Recurrence, Local ; Neoplasm Staging ; Neoplasm, Residual ; Ovarian Neoplasms ; blood ; drug therapy ; pathology ; surgery ; Ovariectomy ; methods ; Remission Induction ; Retrospective Studies ; Survival Rate ; Young Adult

OBJECTIVETo develop an HPLC method for the determination of serum level of Crebanine (Cre) and study on the pharmacokinetics of Cre injection in rabbits.

METHODTo sample blood serum from the rabbits' ears which were injected the Cre by 2.0 mg x kg(-1) at different time and use HPLC to determine the concentration of Cre in it, the pharmacokinetic parameters were accessed by the DAS software.

RESULTCre was fitted to a two compartment open pharmacokinetic model in rabbits. There was no signifiant difference between the male and female rabbits'pharmacokinetic by t-test. The mainly pharmacokinetic parameters were: t1/2alpha = (3. 246 +/-0.222) min, t1/2beta = (36.67+/-5.52) min, Cmax = (1.401 +/- 0.062) mg x L(-1), Vd = (5.928 +/- 0.877) L x kg(-1), Cl = (0. 051 +/-0.003) L x min(-1) x kg(-1).

CONCLUSIONThis experiment can objectively show the pharmacokinetics regularity of Crebanine injection in rabbits. Crebanine injection was a speeding disposition drug (t1/2 <1 h) and disposed extensively and rapidly in rabbits.

Animals ; Aporphines ; administration & dosage ; blood ; pharmacokinetics ; Chromatography, High Pressure Liquid ; Female ; Injections ; Male ; Metabolic Clearance Rate ; Plants, Medicinal ; chemistry ; Rabbits ; Stephania ; chemistry