[PX-12 Promoting Apoptosis of Multiple Myeloma Cell Line H929 Induced by Bortezomib].

OBJECTIVE: To study the effect of PX-12 on apoptosis of multiple myeloma (MM) cell line induced by bortezomib. METHODS: MM cell line H929 cells were divided into PX-12 group, bortezomib group, combination group, and control group. 5.0 µmol/L PX-12, 20 nmol/L bortezomib, combination of the two drugs, and DMSO were given to the above mentioned group, respectively. After culture for 24, 48, and 72 hours, the changes of cell viability were observed, the MM cell activity was detected by MTT method, and the cell cycle distribution and apoptosis of each group was detected by flow cytometry. The intracellular ROS level was measured by H2DCFDA probe labeling. RESULTS: MTT assay showed that after culture for 72 hours, the activity of H929 cells in PX-12 group (P<0.05) and bortezomib group (P<0.01) was significantly lower than that in the control group, while that in the combination group was decreased most significantly (P<0.01). After culture for 48 hours, cells in G1 phase in PX-12 group was decreased to 40%, while cells in S phase and G2/M phase was increased to 28% and 40%, respectively. The cells in bortezomib group also showed a similar distribution after being treated. After treated with PX-12 and bortezomib, the cells in G1 phase were decreased significantly to 19% and 12% in S phase, but increased significantly to 68% in G2/M phase, which was significantly different from PX-12 group and bortezomib group (P<0.01). After culture for 72 hours, the apoptosis rate was 71.3% in the combination group, which was significantly higher than that in PX-12 group, bortezomib group, and control group (20.6%, 33.3%, 10.6%)(P<0.01). After culture for 24 hours, the intracellular ROS level in the combination group was 12015±430.2, which was higher than that in the PX-12 group, bortezomib group, and control group (6729±352.8, 2651±228.3, 1098±164.6, respectively) (P<0.01). CONCLUSION: PX-12 can increase the apoptosis of MM cell line H929 induced by bortezomib, which may be caused by increasing of ROS level.

[Clinical Analysis of Gene Mutation in Adult Patients with B-ALL and Its Influence on Clinical Prognosis].

OBJECTIVE: To investigate the gene mutation in adult patients with B-ALL and its influence on clinical prognosis. METHODS: Clinical data of 226 adult patients with B-ALL were retrospectively analyzed in the period from August 2011 to February 2018. The incidence of gene mutation in all patients were detected, and the influence of mutation gene on clinical prognosis were estimated. Cox regression model were used to evaluate the independent prognostic factors. RESULTS: 208 (92.04%) of 226 patients showed gene mutations, and the median mutation number was 2 (0-8). Among them, 54 cases (23.89%) showed 14 or more mutations. The top five mutation types of all patients were SF1, FAT1, MPL, PTPNII and N-RAS respectively. The median OS and median RFS times of 226 patients were 27.0 (5.5-84.0) months and 22.5 (0-81.0) months respectively. The OS and RFS times of Ph- B-ALL patients with JAK1 and JAK2 mutations were significantly shorter than those of patients without JAK2 mutations (P＜0.05). The OS and RFS times of Ph- B-ALL patients with abnormal JAK-STAT signaling pathway were significantly shorter than those of patients without abnormal JAK-STAT signaling pathway (P＜0.05). The OS and RFS times of Ph+ B-ALL patients with epigenetic related signaling pathway mutations were significantly shorter than those of patients without epigenetic related signaling pathway mutations (P＜0.05). Cox regression model multivariate analysis showed that WBC level was the independent influencing factor for total survival time and relapse-free survival time in adult B-ALL patients (P＜0.05). With or without JAK2 mutation and WBC level were the independent influencing factor for overall survival time and relapse-free survival time of adult Ph- B-ALL patients (P＜0.05). CONCLUSION: Gene mutations are common in all adult B-ALL patients, and the clinical prognosis of patients with JAK and epigenetics-related signaling pathway mutations is worsen, while the WBC level closely relates to the clinical prognosis of the patients.

[ITRAQ Coupled with 2DLC-MS/MS to Screen and Identify Speci-fic Bio-markers of T-Cell Acute Lymphoblastic Leukemia].

OBJECTIVE: To screen and identify potential biomarkers specific for T-cell acute lymphoblastic leukemia (T-ALL). METHODS: Sera were collected from 20 newly diagnosed B-cell acute lymphoblastic leukemia (B-ALL) patients and 20 T-ALL patients. Proteins were extracted, purified and digested with trypsin. All specimens were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) and two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) in a data-dependent mode. Enzyme-linked immunosorbent assay (ELISA) was used to analyze the expression of serum soluble L-selectin (sL-selectin). RESULTS: A total of 468 proteins were identified from distinct peptides. Compared with B-ALL group, 31 proteins were significantly differentially up-regulated while 7 proteins were significantly down-regulated in T-ALL group, sL-selectin was the higher up-regulated in these differential expression proteins. The overexpression of sL-selectin in T-ALL was verified by ELISA. CONCLUSION: There are the differentially expressed proteins between T-ALL and B-ALL, and the sL-selectin is specific for T-ALL, which can not only become a new biomarker for the diagnosis and prognosis of T-ALL, but also can be used as a potential target for therapy of this leukemia.

[Effect of Y chromosomal length variation on male reproductive dysfunction].

OBJECTIVE: To investigate the effects of big and bit Y chromosome configurations on male fertility and to evaluate the relevant clinical significance. METHODS: The relevant cases were divided into A and B groups. Group A included male infertile cases. Group B included cases whose wives had adverse pregnancy history or the abnormal amniotic fluid punctures. The cytogenetics of the patients were examined by culturing peripheral-blood lymphocytes and G-banding technology, and karyotyping analysis techniques were used to study the big and bit Y chromosomes in the two different groups. RESULTS: Among 2 139 cases, 98 cases were found with abnormal karyotype of big and bit Y chromosomes. There was no significant difference in the abnormal rate of the length variation of the Y chromosomal karyotypes between the male infertility group and the adverse pregnancy outcome group. In the male infertile group (group A), there was no significant difference in the abnormal rate between the big Y chromosome and the bit Y chromosome. In the group with adverse pregnancy outcomes (group B), the abnormal rate of the big Y chromosome karyotyping was significantly higher than that of the bit Y chromosome karyotyping. The main clinical effects of groups A and B were azoospermia, oligozoospermia, poor spermia, abortion, embryonic diapause and fetal anomalies, etc . CONCLUSION: The big and bit Y chromosomal abnormality results in not only the male infertility directly, but also an important and continuous reason of adverse pregnancy outcomes, of which the detailed mechanism needs to be further investigated.

[Clinical effect on women with different types of endometriosis related infertility treated by conservative surgery].

OBJECTIVE: To study pregnancy outcome and recurrence in patients with different type of endometriosis related infertility treated by conservative surgery. METHODS: From January 2005 to December 2010, 79 patients with endometriosis related infertility underwent conservative laparoscopic surgery in Peking University First Hospital, including 16 cases with deep infiltrating endometriosis, 39 cases with ovarian endometriosis and 24 cases with peritoneal endometriosis. At 1 to 5 years follow-up after surgery, natural pregnancy outcome and recurrence were studied. RESULTS: (1) The accumulated pregnancy rate were 6/16 in deep infiltrating endometriosis group, 36% (14/39) in ovarian endometriosis group, and 46% (11/24) in peritoneal endometriosis group, which did not reached statistical difference (P > 0.05). (2) The median interval between pregnancy and surgery were 38.5 months in deep infiltrating endometriosis group, 9.5 in ovarian endometriosis group and 6.0 months in peritoneal endometriosis group. The median interval in deep infiltrating endometriosis was significantly longer than that in peritoneal endometriosis group and ovarian endometriosis group (P < 0.01). Total of 11 patients in peritoneal endometriosis group and 11 patients in ovarian endometriosis group acquired pregnancy at 18 months after surgery. (3) The recurrent rate were 5/16 in deep infiltrating endometriosis group, 13% (5/39) in ovarian endometriosis group and 4% (1/24) peritoneal endometriosis group, respectively (P > 0.05). CONCLUSIONS: There was no difference among these three groups in accumulated pregnancy rate. However, the interval between pregnancy and surgery was significantly longer in patients with deep infiltrating endometriosis when compared with those in the other groups.

[Effect of Ras signal regulating Cox7a2 expression and its sub-cellular location in testicular interstitial cell].

OBJECTIVE: To investigate the co-sub-cellular-location of Cox7a2 and Ras. METHODS: Ras and its mutant plasmid were cloned by RT-PCR and sequence analysis. Cox7a2-pEYFP-N1, Ras-pEYFP-N1 and N17-Ras-pEYFP-N1 fluorescent protein vectors were constructed and transfected into TM3 cells. RESULTS: Cox7a2 was located in the mitochondria, but its location was changed by the expression of Ras. When the dominant negative ras was expressed in the cells, the Cox7a2 located into the mitochondria again. CONCLUSION: Cox7a2 mediated testosterone production, which might be at least in part related with the Ras signaling pathway. Ras may be the regulating target and further investigation is needed to make it clear.

[Testicular sperm cryopreservation for male fertility preservation].

OBJECTIVE: To investigate the effectiveness of testicular sperm cryopreservation in male fertility preservation by evaluating the clinical outcome of ICSI cycles with frozen-thawed testicular sperm for azoospermia patients. METHODS: We retrospectively analyzed 96 samples of cryopreserved testicular sperm obtained by testicular biopsy, vasovasostomy (V-V), vasoepididymostomy (V-E) , of which 55 were subjected to 60 ICSI cycles with frozen-thawed testicular sperm. We evaluated the rates of sperm recovery, fertilization, cleavage, transferable and good-quality embryos, clinical pregnancy, pregnancy outcome, and health of the newborns. RESULTS: All the frozen testicular sperm samples were recovered successfully. The rates of fertilization, 2PN fertilization, cleavage, available embryos and good-quality embryos were 77.6, 69.4, 99.4, 84.5 and 40.8%, respectively. There were transferable embryos in all cycles. Fresh embryos were transferred in 52 of the 60 cycles, with the clinical pregnancy rate of 57.7% (30/52), including 19 singletons and 11 twins, and the rates of implantation and miscarriage were 38.7% (41/106) and 3.33% (1/30). Up to the present time, there have been 20 healthy newborns, including 12 boys and 8 girls, and another 13 ongoing pregnancies. No birth defects have been found so far. CONCLUSION: Desirable clinical outcomes can be obtained from ICSI cycles with frozen-thawed testicular sperm, and testicular sperm cryopreservation is an effective method of fertility preservation for azoospermia males.

[Cesarean scar pregnancy: an analysis of 100 cases].

OBJECTIVE: To explore the clinical diagnosis and treatment of cesarean scar pregnancy (CSP). METHODS: The clinical data of 100 CSP patients during the period of January 2003 to March 2011 were collected for a retrospective analysis. RESULTS: Among 100 cases of CSP, there were cases of asymptomatic (45%, n = 45), vaginal hemorrhage (55%, n = 55) and lower abdominal pain (7%, n = 7); among first diagnosed (n = 81), the cases were diagnosed (n = 75) or misdiagnosed (n = 6); among 19 hospital referrals, 18 were confirmed and 1 case was misdiagnosed as choriocarcinoma. Treatments included ultrasound monitoring curettage after UAE (uterine artery embolism) (n = 56), pure ultrasound monitoring curettage (n = 30), laparoscopic lumpectomy after UAE (n = 2), laparoscopic lumpectomy (n = 2), methotrexate only (n = 3), hysterectomy (n = 2), UAE hemostasis (n = 3) and Foley catheter balloon compression hemostasis (n = 2). No significant difference was found in the average duration of pregnancy, average operative hemorrhage volume and operative duration between 2 management groups of curettage after UAE or pure curettage (P > 0.05). But sac diameter and serum level of ß-HCG were obviously less in the pure curettage group than those in the UAE group. And the distance between gestation sac and bladder significantly was greater than that in the UAE group; in the pure curettage group, 96.7% existed as an endogenous type and only 36.7% yielded small flow signals. One hundred patients recovered before discharge. A follow-up of patients with curettage after UAE (n = 43) and pure curettage (n = 26) had similar recovery of menstruation. Eight cases (4 in each) were pregnant again during the follow-up. One case of recurrent CSP was treated with curettage after methotrexate. CONCLUSION: Early diagnosis and early treatment remain the key for a successful treatment of CSP. Color Doppler ultrasound is important in its early diagnosis and treatment. Different therapeutic modalities may be selected according to specific patient conditions.

[Expression of CIITA Gene in five human cell lines and its significance].

The objective of study was to investigate the relationship between expressions of CIITA and MHC molecules in five human cell lines. The expressions of MHC molecules and CIITA protein were detected by Western blot, immunohistochemistry and flow cytometry. The expression of CIITA gene was measured by RT-PCR. The results indicated that the expression of MHC-II molecules in 5 human cell lines was consistent with expression of CIITA. The cell lines constitutively expressed CIITA also expressed MHC-II molecules, the expression of MHC-II molecules in cell lines expressed CIITA after induction with IFN-gamma also recovered; the cell lines unexpressed CIITA after induction with IFN-gamma did not respond to IFN-gamma-promoting expression of MHC-II molecules. It is concluded that some cell lines cannot express MHC-II molecules which may be related with deficiency of CIITA expression. It suggest that CIITA participates in regulation of MHC-II molecule expression, which may plays a certain role in escape from carcinogenesis under surveillance of immune system.

[Expression of iron regulatory protein-2 and ferritins in intestinal mucosa of rats with iron deficiency].

In order to investigate the influence of iron deficiency on the mRNA expression of iron regulatory protein (IRP(2)) mRNA and ferritins (FN) in intestinal mucosa of rat, the animal model of rat with nutritional iron deficiency was established. According to the measurement of serum iron (sI), serum fertitin (sFn) and Hb, the experiments were divided into 4 groups: control group, recessive iron deficiency group, mild iron deficiency group and moderate iron deficiency group. sI was measured by flame assay and sFN was measured by radioimmunoassay, the expressions of irp(2) mRNA and fn mRNA were detected by RT-PCR. The results showed that (1) with aggravation of iron deficiency, the levels of sI and sFN in experimental groups decreased and had significant difference from that in control group, except sI level in the recessive iron deficiency group; (2) with aggravation of iron deficiency, the expression of irp(2) mRNA in duodenum mucosa elevated, and the expressions of irp(2) mRNA in moderate iron deficiency group and mild iron deficiency group were higher than that in control group (p < 0.01), the expression of irp(2) mRNA in moderate iron deficiency group was higher than that in recessive iron deficiency group (p < 0.05), but the expression of irp(2) mRNA did not showed statistical difference between mild iron deficiency group and moderate iron deficiency group (p > 0.05); (3) with aggragation of iron deficiency, the expression of fn mRNA in dudemum mucosa decreased, the expression levels fn mRNA in control and moderate groups were highest and lowest, respectively, there were significant differences between experimental and control groups (p < 0.05), and between experimental groups (p < 0.05); (4) the expression of irp(2) mRNA and fn mRNA in moderate iron difficiency group showed negative correlation (r = 0.662, p < 0.05). It is concluded that IRP(2) protein serves as an important regulator of iron metabolism in the human body, and regulates iron uptake from the intestine by controlling the expression of fn mRNA at the post transcriptional level.

[Identification of IgG subclass and FVIII binding epitope of an acquired FVIII inhibitor in a bullous pemphigoid patient].

OBJECTIVE: To identify the clinical and laboratory diagnosis of a bullous pemphigoid patient with acquired hemophilia A (AH-A). To identify FVIII binding epitope and IgG subclass of the FVIII inhibitor, and explore the molecular mechanism for AH-A pathogenesis. METHODS: Plasma FVIII activity( FVIII: C) was determined by one-stage assay, the titre of FYIII inhibitor by Bethesda Unit (BU). IgG purification of patient plasma or normal pooled plasma was finished by protein A-agarose column chromatography. Activated partial thromboplastin time (APTT) was assayed for uncovering FVIII inhibitor effect on FVIII in vivo. Combined Western blot analysis by anti-IgG1, IgG2, IgG3 and IgG4 antibodies was used to determine the relative concentration of patient' s IgG subclass. IgG subclass concentrations were quantified by nephelometric method. Solid-phase binding assay of FVIII and FVIII inhibitor, combined with Western blot was used to recognize the binding epitope at which the FVIII inhibitor bound to FVIII. RESULTS: (1) Plasma APTT value of patient was prolonged evidently and could not be corrected by normal pooled plasma. Patient's FVIII: C was < 1.5%. The titre of FVIII inhibitor in patient plasma was 147.8 BU. (2) The purified patient IgG was able to inhibit FVIII: C of normal pooled plasma significantly with a dose dependent manner, and the patient plasma could prolong rabbit plasma APTT markedly with a time dependent manner. (3) The FVIII inhibitor was predominantly then of IgG4 subtype with a minority IgG1, and the concentration of IgG4 and IgG1 in the patient was higher than that in normal. The FVIII inhibitor reacted with FVIII 44 x 10(3) fragment epitope. CONCLUSIONS: The inhibiting effect of FVIII inhibitors on FVIII: C in the bullous pemphigoid patient with AH-A is determined and the IgG subclass of the FVIII inhibitor is identified. A binding epitope for the FVIII inhibitor is a FVIII 44 x 10(3) fragment. The results provides evidence for understanding the pathogenesis of AH-A.