UCHL-3 as a potential biomarker of ovarian cancer.

OBJECTIVE: This study explored promising prognostic and immune therapeutic candidate biomarkers for OC and determined the expression, prognostic value, and immune effects of UCHL3. METHODS: UCHL3 expression and clinical data were investigated using bioinformatic analysis. CCK8 and transwell assays were conducted to evaluate the impact of UCHL3 on proliferation and migration, and the effects of UCHL3 were further validated in a mouse model. Univariate and least absolute shrinkage and selection operator regression analyses were performed to construct a novel UCHL3-related prognostic risk model. Gene set enrichment analysis (GSEA) and immune analysis were performed to identify the significantly involved functions of UCHL3. Finally, bioinformatic analysis and immunohistochemistry were performed to explore the effect of UCHL3 on chemotherapy. RESULTS: UCHL3 expression was upregulated and associated with worse overall survival (OS) in OC. UCHL3 depletion repressed cell proliferation and migration both in vitro and in vivo. Furthermore, 237 genes were differentially expressed between the high and low UCHL3 expression groups. Subsequently, a UCHL3-related prognostic signature was built based on six prognostic genes (PI3, TFAP2B, MUC7, PSMA2, PIK3C2G, and NME1). Independent prognostic analysis suggested that age, tumor mutational burden, and RiskScore can be used as independent prognostic factors. The immune infiltration analysis and GSEA suggested that UCHL3 expression was related to the immune response. In addition, UCHL3 expression was higher in platinum-resistant OC patients than in platinum-sensitive patients. UCHL3 overexpression was associated with poorer OS. CONCLUSION: UCHL3 overexpression contributes to aggressive progression, poor survival, and chemoresistance in OC. Therefore, UCHL3 may be a candidate prognostic biomarker and potential target for controlling progression and platinum resistance in OC.

A label-free and real-time method for evaluating cytotoxicity of CD19 CAR-T cells / 医学研究生学报

ObjectiveUsing Chromium-51 release assay, lactate dehydrogenase release assayand other methods to detect the cytotoxicity of CD19 CAR-T cells is cumbersome, with low repeatability and poor stability. This study aims to establish a label-free and real-time method for detectingspecific cytotoxicity of CD19 CAR-T cells.MethodsIn order to establish target cell models for cytotoxic assay of CD19 CAR-T cells by using Real Time Cellular Analysis (RTCA) system,the adherent human breast cancer cells were infected with lentiviral vectors encoding CD19. CD19 expression on the transduced cells was detected by flow cytometry. The cellsexpressing CD19 stably werethen sorted by fluorescence activated cell sorting (FACS).With such cells as target cells, CD19 CAR-T cells and BCMA CAR-T cells as effector cells, RTCAsystem was used to evaluate the cytotoxicity of CAR-T cells against target cells.ResultsMDA-MB-231 and SKBR3cells with stable expression CD19were obtained in this study.The results of flow cytometry showed that positive expression rate of CD19 in MDA-MB-231/CD19 cells and SKBR3/CD19 monoclonal cells were 99.03% and 98.91%,respectively.RTCA results showed that with MDA-MB-231 and MDA-MB-231/CD19 cells as target cells,CD19 CAR-T cells showed significant cytotoxicity to MDA-MB-231/CD19 cellsat the effector-target ratio of 5∶1, 1∶1 and 1∶5,but not to MDA-MB-231 cells. With SKBR3 and SKBR3/CD19 cells as target cells, CD19 CAR-T cells showed significant cytotoxicity to SKBR3/CD19 cellsat the effector-target ratio of 5∶1and 1∶1. When the effector-target ratio was 1∶5, there was no obvious cytotoxicity.The data of MDA-MB-231/CD19 or SKBR3/CD19 as target cells and CD19 CAR-T as effector cells were analyzed separately, showing that when the number of target cells was the same, the cytotoxicity detected by RTCA increased as the number of CD19 CAR-T cells increased.The cytotoxic assays of CD19 CAR-T cells showed specificity and dose-response relationship of CD19 CAR-T cytotoxicity against the target cells.ConclusionThis study established a method for evaluating cytotoxicity of CD19 CAR-T cells that is real-time, label-free, simple and convenient.

ADRB2 Gene Knockout in Human Primary T Cells by Multiple sgRNAs Construced using CRISPR/Cas9 Technology / 中国实验血液学杂志

OBJECTIVE@#To knockout ADRB2 gene rapidly and efficiently in human primary T cells by using CRISPR/Cas9 technology and multiple sgRNAs strategy.@*METHODS@#Six paired-sgRNAs, which were designed to target the 5' constitutive coding exons of ADRB2 gene, were cloned into pGL3-U6-sgRNA-PGK-Puro vector separately. The expre-ssion vectors containing the single sgRNAs were constructed and transiently co-transfected into HEK-293T cell line with Cas9 expression vector. The sgRNA-mediated cleavage efficiency was tested by T7EN I digestion assay. Concatenating four highly efficient paired sgRNAs were cloned into pGL3-U6-sgRNA-ccdB-EF1α-Puro expression vector. The reco-mbinant plasmid allows the cells to express 4 sgRNAs, which target different sites on the ADRB2 genomic locus. The cleavage efficiency and mutation model were tested by T7EN I digest assay and T-A cloning technique. Multiple sgRNAs plasmid and Cas9 plasmid was transiently transferred into human primary T cells by electroporation. Flow cytometry (FCM) was used to detect the knockout efficiency of β2 adrenergic receptor (β2-AR).@*RESULTS@#The results of T7EN I digestion and TA cloning sequencing showed that the multiple sgRNAs strategy could obtain more abundant mutation types and higher gene editing efficiency than single sgRNA. In addition to the deletion and insertion of bases, large fragment DNA deletions and inversions could be observed. All of the random 10 TA clones for detection were genetically modified, thus the mutation efficiency was as high as 100%. FCM assay showed that 43.09% of the cells in the control T cells were β2-AR positive, but the proportion of β2-AR positive cells in the multiple sgRNAs electrotransformed T cells decreased to 25.61%.@*CONCLUSION@#A method, which is simple and operable, for knocking out β2-AR in human primary T cells has been established preliminarily. The results are helpful for the further study of the role of β2-AR in human T cells.

A comparative study of high-viscosity cement percutaneous vertebroplasty vs. low-viscosity cement percutaneous kyphoplasty for treatment of osteoporotic vertebral compression fractures / 华中科技大学学报（医学）（英德文版）

The clinical effects of two different methods-high-viscosity cement percutaneous vertebroplasty (PVP) and low-viscosity cement percutaneous kyphoplasty (PKP) in the treatment of osteoporotic vertebral compression fractures (OVCFs) were investigated. From June 2010 to August 2013, 98 cases of OVCFs were included in our study. Forty-six patients underwent high-viscosity PVP and 52 patients underwent low-viscosity PKP. The occurrence of cement leakage was observed. Pain relief and functional activity were evaluated using the Visual Analog Scale (VAS) and Oswestry Disability Index (ODI), respectively. Restoration of the vertebral body height and angle of kyphosis were assessed by comparing preoperative and postoperative measurements of the anterior heights, middle heights and the kyphotic angle of the fractured vertebra. Nine out of the 54 vertebra bodies and 11 out of the 60 vertebra bodies were observed to have cement leakage in the high-viscosity PVP and low-viscosity PKP groups, respectively. The rate of cement leakage, correction of anterior vertebral height and kyphotic angles showed no significant differences between the two groups (P>0.05). Low-viscosity PKP had significant advantage in terms of the restoration of middle vertebral height as compared with the high-viscosity PVP (P<0.05). Both groups showed significant improvements in pain relief and functional capacity status after surgery (P<0.05). It was concluded that high-viscosity PVP and low-viscosity PKP have similar clinical effects in terms of the rate of cement leakage, restoration of the anterior vertebral body height, changes of kyphotic angles, functional activity, and pain relief. Low-viscosity PKP is better than high-viscosity PVP in restoring the height of the middle vertebra.

Construction of an oncolytic adenovirus expressing small hairpin RNA and targeting the SATB1 gene / 中华男科学杂志

<p><b>OBJECTIVE</b>To construct an oncolytic adenovirus with the DD3 promoter regulation, expressing small hairpin RNA and targeting the SATB1 gene (SATBI-shRNA), and to evaluate its potential for inhibiting the growth of human prostatic carcinoma cells (LNCaP) in vitro.</p><p><b>METHODS</b>SATB1-shRNA expression cassettes containing the H1 promoter were produced by PCR from pSilencer3. 1-SATB1 and inserted into the pZD55 vector, and the recombinant plasmid pZD55-SATB1-shRNA was constructed, pZD55SATB1-shRNA and pZXC2-DD3-E1A were digested with EcoRV and Xba I , and the obtained expression cassettes linked each other to construct the recombinant plasmid pDD3-ZD55-SATB1, which was cotransfected with the pBHGE3 packaging plasmids mixture into 293 cells by Effectence. The recombined adenoviruses DD3-ZD55-SATB1 were identified by PCR. Viruses were propagated on HEK293 cells and purified by standard techniques, and the functional PFU titers determined by plaque assay on 293 cells. The antitumor potential of DD3-ZD55-SATB1 to LNCaP was evaluated by the crystal violet dye method. The expression level of the E1A gene was detected by Western blot, and that of the SATB1 gene in the adenovirus-infected LNCaP cells by both Western blot and RT-PCR.</p><p><b>RESULTS</b>An oncolytic adenovirus expressing SATB1-shRNA with the DD3 promoter regulation, DD3-ZD55-SATB1, was constructed successfully, whose functional PFU titer was 1.2 x 10(10) PFU/ml. DD3-ZD55-SATB1 showed an obvious cytopathic effect and a selective expression of E1A in the adenovirus-infected LNCaP cells; it exhibited a high LNCaP-targetability and specific SATB1-silencing effect.</p><p><b>CONCLUSION</b>The successful construction of the oncolytic adenovirus DD3-ZD55-SATB1 offers a new tool for researches on the gene therapy for human prostate cancer.</p>

Adriamycin enhances the sonodynamic effect of chlorin e6 against the proliferation of human breast cancer MDA-MB-231 cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the effect of adriamycin (ADM) in enhancing the sonodynamic effect of chlorin e6 against the proliferation of human breast cancer MDA-MB-231 cells in vitro.</p><p><b>METHODS</b>MDA-MB-231 cells were treated with ultrasound/Chlorin e6 alone or in combination with ADM, and the changes in the cell proliferation was determined by MTT assay.</p><p><b>RESULTS</b>Ultrasound (1.0 MHz) at the power intensity of 0.5-2.0 W/cm2 inhibited the proliferation of MDA-MB-231 cells in an intensity-dependent manner, and chlorin-e6 (0.05-1.6 mg/ml) and ADM (0.1-0.4 g/ml) alone both inhibited the proliferation of MDA-MB-231 cells dose-dependently. Compared with ultrasound (0.5 W/cm2, 1.0 MHz, 60 s) or chlorin-e6 (0.05-0.2 mg/ml) alone, a combined treatment with ultrasound and chlorin e6 significantly enhanced the inhibitory effect on the proliferation of MDA-MB-231 cells (P<0.05). ADM significantly enhanced the sonodynamic effect of chlorin e6 (0.1 mg/ml) against the cell proliferation of MDA-MB-231 cells (P<0.05), and the effect was schedule-dependent, which was greater when ADM was added after the sonodynamic treatment (P<0.05).</p><p><b>CONCLUSION</b>ADM can enhance the sonodynamic effect of chlorin e6 against the proliferation of MDA-MB-231 cells in vitro.</p>

Skip metastasis of prostate cancer: diagnosis and treatment / 中华男科学杂志

<p><b>OBJECTIVE</b>To improve the diagnosis and treatment of far advanced prostate cancer without clinically detectable bone metastasis.</p><p><b>METHODS</b>Cancer metastatic lesions were found in the liver and lungs respectively of two patients on routine medical examination, and only an abnormally elevated level of the serum prostate specific antigen (PSA) was observed in the following system examinations. The patients were diagnosed as having prostate cancer by prostate biopsy. MRI showed a discontinued prostate capsule, and ECT revealed no bone metastasis. Diagnostic treatment was conducted by giving LHRHa combined with antiandrogens. One of the patients underwent surgical castration at 12 months, and both received intensity modulated radiation therapy (80 Gy) at 15 and 18 months, respectively.</p><p><b>RESULTS</b>The metastatic lesions in the liver and lungs of the patients were either absent or significantly reduced after treated by maximal androgen blockade for 3 months, and all disappeared after 6 months'treatment, with the PSA level stabilized at less than 0.02 microg/L in one patient, and around 0.5 microg/L in the other. Antiandrogen treatment was suspended after radiotherapy. The results of liver, lung and bone scanning were normal during the 12-month follow-up, and the PSA level was below 1.0 microg/L.</p><p><b>CONCLUSION</b>Remote metastasis of prostate cancer may occur in ectosteal organs first, which deserves special attention. A combination of different treatment methods promises satisfactory results.</p>

[In vitro expansion and function of cord blood megakaryocyte].

OBJECTIVE: Cord blood (CB) provides a rich source of stem cells for transplantation. CB transplantation has been used widely after myeloablative therapy. One major disadvantage of CB transplantation is delayed platelet engraftment. The aim of this study was to hasten platelet engraftment by investigating the ability of different hematopoietic growth factor combinations to generate large numbers of megakaryocyte (Mk) from CB and by evaluating the biologic characteristics and function of the expanded Mk. METHODS: CB samples were obtained at the end of normal full-term deliveries with informed consent. Mononuclear cells (MNCs) were isolated from CB using Ficoll density centrifugation. MNC population was positively selected for CD(34) expression by magnetic cell sorting (MACS). CD(34)(+) cells were cultured in serum-free and stroma-free medium containing the following two different cytokine combinations: thrombopoietin (TPO) + stem cell factor (SCF) + interleukin (IL) -3 + IL-6 and TPO + SCF. Cultures were characterized after 3, 7, 10 and 14 days by flow cytometry, colony forming unit-megakaryocyte (CFU-Mk) and maturation evaluation (Mk ploidy). The expanded Mk function was examined by the platelet activation in vitro and severe combined immunodiffiency (SCID) mice transplantation in vivo. RESULTS: Different results were observed with different culture conditions. With the first cytokine combination optimal expansion of CD(41)(+) cells was observed on day 10, but the optimal expansion of Mk progenitors (CD(34)(+)CD(41)(+)) was observed on day 7, with a median 121 and 44-fold increase at the starting cell dose. This result was also proven by CFU-Mk. The largest numbers of CFU-Mk were also observed on day 7. The degree of maturation of Mk cells also increased as suggested by DNA content of CD(41)(+) cells, which means that CD(34)(+) cells cultured for 3 - 7 days were richer in primitive Mks, while those cultured for 10 - 14 days had greater numbers of more differentiated Mks. For the second cytokine combination, CD(41)(+) and CD(34)(+)CD(41)(+) cells were fewer than the first one, but it produced 36 and 85-fold CD(34)(+)CD(41)(+) and CD(41)(+) respectively on day 7. Platelet activation test confirmed that the expanded Mks had normal function. Therefore, the expanded Mks could be transplanted into the SCID mice bone marrow and produce human platelet in the peripheral blood of the mice. CONCLUSION: Ex vivo expanded Mk might facilitate CB transplantation and help shorten the period of post-transplant thrombocytopenia.

In vitro expansion and function of cord blood megakaryocyte / 中华儿科杂志

<p><b>OBJECTIVE</b>Cord blood (CB) provides a rich source of stem cells for transplantation. CB transplantation has been used widely after myeloablative therapy. One major disadvantage of CB transplantation is delayed platelet engraftment. The aim of this study was to hasten platelet engraftment by investigating the ability of different hematopoietic growth factor combinations to generate large numbers of megakaryocyte (Mk) from CB and by evaluating the biologic characteristics and function of the expanded Mk.</p><p><b>METHODS</b>CB samples were obtained at the end of normal full-term deliveries with informed consent. Mononuclear cells (MNCs) were isolated from CB using Ficoll density centrifugation. MNC population was positively selected for CD(34) expression by magnetic cell sorting (MACS). CD(34)(+) cells were cultured in serum-free and stroma-free medium containing the following two different cytokine combinations: thrombopoietin (TPO) + stem cell factor (SCF) + interleukin (IL) -3 + IL-6 and TPO + SCF. Cultures were characterized after 3, 7, 10 and 14 days by flow cytometry, colony forming unit-megakaryocyte (CFU-Mk) and maturation evaluation (Mk ploidy). The expanded Mk function was examined by the platelet activation in vitro and severe combined immunodiffiency (SCID) mice transplantation in vivo.</p><p><b>RESULTS</b>Different results were observed with different culture conditions. With the first cytokine combination optimal expansion of CD(41)(+) cells was observed on day 10, but the optimal expansion of Mk progenitors (CD(34)(+)CD(41)(+)) was observed on day 7, with a median 121 and 44-fold increase at the starting cell dose. This result was also proven by CFU-Mk. The largest numbers of CFU-Mk were also observed on day 7. The degree of maturation of Mk cells also increased as suggested by DNA content of CD(41)(+) cells, which means that CD(34)(+) cells cultured for 3 - 7 days were richer in primitive Mks, while those cultured for 10 - 14 days had greater numbers of more differentiated Mks. For the second cytokine combination, CD(41)(+) and CD(34)(+)CD(41)(+) cells were fewer than the first one, but it produced 36 and 85-fold CD(34)(+)CD(41)(+) and CD(41)(+) respectively on day 7. Platelet activation test confirmed that the expanded Mks had normal function. Therefore, the expanded Mks could be transplanted into the SCID mice bone marrow and produce human platelet in the peripheral blood of the mice.</p><p><b>CONCLUSION</b>Ex vivo expanded Mk might facilitate CB transplantation and help shorten the period of post-transplant thrombocytopenia.</p>

The value of pre-operative semen analysis as a restore index of fertilizing capacity after varicocelectomy / 中华男科学杂志

<p><b>OBJECTIVE</b>To discuss the value of pre-operative semen analysis of patients with varicocele as a predictive restore index of sperm motility and fertilizing capacity after varicocelectomy.</p><p><b>METHODS</b>Semen analysis was carried out with computer-aided sperm analyzer in 107 patients with varicocele and all patients were referred to the clinic with diagnosis of male infertility. Stratification of patients as group A (n = 32), B ( n = 36) and C (n = 39) was based on pre-operative total motile sperm count (TMSC). Follow-up included semen analysis and pregnancy data after three months following left or bilateral varicocelectomy.</p><p><b>RESULTS</b>The average post-operative TMSC increased significantly when compared with the pre-operative. However, a mean absolute increase in group A and B was better than that in group C (P < 0.05). Of the 68 patients in groups A and B based on pre-operative TMSC, 56 patients' TMSC (82.4%) was > or =20 x 10(6) after varicocelectomy, and that of only 8 (20.5%) patients in group C was > or =20 x 10(6) following varicocelectomy. Of the 98 patients wives, 36 had natural conception. Pregnancy rates in groups A and B were higher than that in group C (P < 0.05).</p><p><b>CONCLUSION</b>Varicocelectomy may be the most effective method to patients with varicocele with pre-operative TMSC > or = 5 x 10(6), but it may be not the best method for patients with severe oligoasthenospermia (pre-operative TMSC < 5 x 10(6)).</p>

Effects of siRNA against Ki67 gene on the proliferation and apoptosis of human renal carcinoma cells / 中华泌尿外科杂志

Objective To evaluate the effects of small interfering RNA(siRNA)against Ki67 gene on the proliferation and apoptosis of human renal carcinoma cell line 786-0 cells.Methods The human renal carcinoma 786-0 cells were treated with Ki67-siRNA(100 nmol/L).The mRNA expression of Ki67 was detected by RT-PCR.The protein expression of Ki67 was detected by Western blot and immunohisto- chemical technique,respectively.The proliferation of 786-0 cells was detected by MTT assay.The apoptosis of 786-0 cells was detected by TUNEL assay.Results RT-PCR and Western blot analysis showed that the Ki67 mRNA and Ki67 protein expression levels of the 786-0 cells treated with Ki67-siRNA were(37.6?1.9)% and(46.4?0.9)% ,respectively,which were significantly lower than those of controls [(97.3?0.9)% and(95.3?0.9)%,P＜0.01],The Ki67 positive expression rate of 786-0 cells treated with Ki67-siRNA by immunohistochemical technique was 52.5?2.3,which was significantly lower than that of controls(114.5?4.9 ,P＜0.01).The proliferation-inhibiting rate and apoptosis rate of the 786-0 cells trea- ted with Ki67-siRNA were( 63.6?1.6)% and(41.7?0.6)% ,respectively,which were significantly higher than those of controls [(2.8?0.2)% and(10.3?1.4)%,P＜0.01].Conclusions siRNA against Ki67 gene can inhibit the proliferation and induce the apoptosis by blocking Ki67 expression of hu- man renal carcinoma 786-0 cells.The inhibition of Ki67 expression by siRNA may be a promising approach in gene therapy for renal cancer.