Objective To study risk factors of cardiovascular disease (CVD) and status of bone mineral density (BMD) in women with hypoestrogenism.Methods From Jul 2011 to April 2013,a total of 256 women with hypoestrogenism in the First Affiliated Hospital of Shanxi Medical University were enrolled in this retrospective study,which were divided into four groups:133 women in ppausal group,25 women in premature ovarian failure (POF) group,67 women in menopausal transition group and 31 women in premature ovarian failure transition group.General statue,CVD risk factors and BMD were compared among four groups.General statue include menopausal period,menopausal symptoms (Kupperman Index),CVD risk factors include body mass index,blood pressure,waist circumference,waist-hip ratio,blood lipids and glucose,BMD include left hip,lumbar spine bone mineral density and T or Z value.Results (1) The median menopausal period were 3.4 years in postmenopausal group and 3.6 years in premature ovarian failure group,which did not show no statistical difference (P ＞ 0.05).Kupperman Index in four groups were 12 in postmenopausal group,9 in POF group,9 in menopausal transition group and 8 in premature ovarian failure transition group,which reached statistical difference (P ＜ 0.05).(2) The difference of body mass index (BMI),waist circumference,waist-hip ratio,diastolic blood pressure were no statistically significant among four groups(P ＞ 0.05) ; the systolic blood pressure in four groups were 120,110,110,110 mm Hg (1 mm Hg =0.133 kPa),their differences were statistically significance (P ＜ 0.05); the high-density lipoprotein (HDL-C) was 1.6 mmol/L in postmenopausal group,and 1.3 mmol/L in premature ovarian failure transition group,their differences were all statistically significance (P ＜ 0.05) ; the difference of the fasting plasma glucose (FPG) was not statistically different in 4 groups (P ＞0.05).(3) The abnormal rate of lower bone mass in lumbar spine were 57％ (46/81) postmenopausal group,8/15 in POF group,32％ (9/28) in menopausal transition group,12/19 in premature ovarian failure transition group,and osteoporosis was 9％ (7/81),3/15,1％ (3/28)and 0 respectively,their differences were statistically different (P ＜ 0.05) ; the abnormal rate of BMD of left hip and lumbar spine of 11/15 and 12/16 in POF group was higher than 65％ (53/81) in postmenopausal group.In the mean time,the abnormal rate of BMD of left hip and lumbar spine were,12/19 and 10/20 in premature ovarian failure transition group,which were significantly higher than 43％ (12/28) and 39％ (12/31) in the menopausal transition group.Conclusions The menopausal symptoms resulting from hypoestrogenism in natural postmenopausal women are mostly remarkable.The decrease of BMD in lumbar spine is more significant than that of left hip among postmenopausal women.Women with earlier menopause was prone to cause the changes of blood fat and abnormal of BMD,especially HDL-C decreased significantly compared with those natural postmenopause,it is more likely to cause CVD and osteoporosis.

To produce monoclonal antibody (mAb) specifically against human thrombomodulin (hTM), an immune-tolerizing procedure was employed to generate monoclonal antibodies specific to hTM. Female BALB/c mice were first immunized with CHO cells following at 10 min, 24 h, 48 h by intraperitoneal injection of different doses of cyclophosphamide (CP) 2 times at an interval of 2 weeks, thereby tolerizing the mice to common epitopes shared between CHO and CHO-TM5 cells. Subsequently the selected mice with the lowest titer of serum polyclonal antibody by cellular enzyme-linked immunoabsorbent assay (CELISA) were immunized with CHO-TM5 cells, which have stable high level expression of hTM, to produce antibodies specific to hTM 3 times at an interval of 2 weeks. On the third day after the third immunization, mouse with the highest titer of serum polyclonal antibody was sacrificed and spleen cells were harvested to prepare hybridoma cells with SP2/0 cells at the ratio of 10 to 1. Hybridoma cells were then cultured at 96 well plates for screening with CELISA. To improve probability to obtain specific mAb, CELISA was applied twice. The first CELISA was done with polyethylene ELISA plate with a monolayer of CHO-TM5 cells. The positive clones from the first screen were then selected by reacting with similar screening ELISA plate but having CHO cells monolayer instead. Only clones that were positive for the first screening and negative for the second screening were kept, and called as CHO-TM5 +CHO- hybridoma cells. BALB/c mice were intraperitoneally injected with the selected hybridoma cells. Ascites were collected and monoclonal antibodies were purified using FPLC, and its Ig class, subclass, and titer were then determined respectively. The specificity of the yielded mAb was identified with CELISA, flow cytometry, ABC immunohistochemistry and immunoblotting. Detection of CELISA showed that 100 mg/kg dose of CP could tolerize the mouse to common epitopes shared between CHO and CHO-TM5 cells. And CELISA also discovered that all hybridomas positive for CHO-TM5 cells were negative for CHO cells. Five lines of positive hybridoma cells had been obtained altogether and 2F7 was selected randomly for next investigation. The Ig subclass of the mAb 2F7 was IgG1 and the titer of ascitic mAb was 1?10-6. Furthermore, the content of ascitic mAb was 19.56 g/L and chromosome numbers is 98. Flow cytometry, CELISA and Western blotting assays demonstrated that mAb 2F7 could specifically recognize hTM expressed on CHO-TM5 and human umbilical vascular endothelial cells (HUVEC). Meanwhile, the tissue specificity of mAb 2F7 was also identified by immunohistochemical ABC staining. On the other hand, Western blotting assays indicated that mAb 2F7 could recognize the antigen protein with 105 ku molecular mass under reduction condition. Moreover, the dissociation constant of mAb 2F7, 1.22? 10-9 mol/L, indicated the affinity higher than some others. The results suggest that the immunotolerizing protocol provides a convenient general method for producing antibodies specific to desired protein isoforms. mAb 2F7 can specifically recognize the natural hTM expressed mainly on vascular endothelial cells, which will potentially useful for investigating the functions and clinic values of hTM.

Objective To construct the recombinant pMD19-exon18-exon20 plasmid containing locus G719S and T790M of EGFR gene associated with cervical cancer,which may provide a template for preparing the mutant recombinant vector of EGFR gene.Methods Using the healthy human genome DNA as templates,the segments of exon 18 and exon 20 of EGFR gene were amplified by two pairs of specific primers which were designed based on the sequences of overlapping and complementary area.The amplified segments were linked by overlap PCR.The products of linked exon18-exon20 were further inserted into the vector pMD19-T.The constructed recombi nant pMD19-exon18-exon20 plasmid was finally transformed into competent cells E.coli DH5α and then identified by PCR with bacterial solution and genome sequencing.Results The amplified fragments of exon18 and exon20 were clearly appeared at 778 bp and 596 bp and the fused product of exon18-exon20 was showed at 1 374 bp on agarose gel electrophoresis.The recombinant plasmid of fusion EGFR gene was consistent with the expected results via bacterial PCR assay and DNA sequencing.Conclusion We successfully fused the segments of exon18 with exon20 and constructed the recombinant expression vector of EGFR gene by using overlap PCR method.

Objective To construct mutant recombinant vector of epidermal growth factor receptor ( EGFR) gene G719S and T790M sites associated with cervical cancer, lay the foundation for the detection of EGFR gene mutation in cervical cancer. And using it to es-tablish a molecular switch platform to detect cervical cancer EGFR gene mutations. Methods Using the wild-type recombinant plasmid as template, the mutant fusion target fragment were amplified by overlap PCR, then connect this target fragment into the vector pMD19-T. The constructed mutant recombinant plasmid was finally transformed into competent cells E.coli DH5αfurther identified by PCR with bacterial solution and genome sequencing. Establishing the molecular switch for the detection of clinical cervical cancer samples. Re-sults The G719S and T790M mutations were successfully certified by genome sequencing, and the site-directed mutant vector was successfully constructed. In addition, a molecular switch detection platform was also successfully established for the detection of cervical cancer tissue DNA. Conclusion We successfully constructed an EGFR gene mutant recombinant vector by overlap PCR technique, which providing a new technical means for gene site-directed mutagenesis. And the molecular switch detection platform was successfully established based on it, which furnishing a new method for clinical detection of EGFR gene mutations.

Endometrial cancer originates from the endometrium and is one of the common gynecologic malignancies, with its incidence and mortality rate increasing year by year. Although endometrial cancer is more prevalent in the peri- and postmenopausal female population, it has been an evident trend in recent years towards younger patients. For young patients who have not yet given birth but intend to do so, the application of progestins in endometrial cancer treatment has made significant progress in clinical practice. Considering the existence of large individual differences and unclear mechanisms of action in the clinical application of progestins, this paper aims to provide an overview of the current clinical application status, efficacy, hormone resistance, and its mechanisms in the context of hormone therapy.

Objective:To explore the key points of the pathological and differential diagnoses of extra-medullary masses of hematopoietic cell tumors of ambiguous lineage, and to discuss the possible solutions.Methods:Five hematopoietic cell tumors of ambiguous lineage cases were collected, including myeloid sarcoma, mixed phenotype acute leukemia, B/myeloid, T-lymphoblastic lymphoma combined with acute myeloid leukemia, acute undifferentiated leukemia with cutaneous MPDCP and early T-precursor cell acute lymphoblastic leukemia. The data including morphology, immunostaining, and flow cytometry analysis were collected, and we explored the problems and differential diagnosis in the diagnosis of hematopoietic cell tumors of ambiguous lineage.Results:The five cases showed that the accurate pathological diagnosis and classification of hematopoietic cell tumors of ambiguous lineage should be based on lineage-specific antigens. Moreover, tumor cells have the potential of multi-directional differentiation. In different sites or different periods, the differentiation of tumor cells may be different. Biopsy and detection of all related markers should be performed for the initial diagnosis, and the detection should be repeated when the condition of the patient changes. Combined application of multi-techniques, including morphology and flow cytometry analysis, is recommend for the diagnosis of hematopoietic cell tumors of ambiguous lineage, since the conventional morphology and immunophenotyping methods are limited.Conclusion:Hematopoietic cell tumors of ambiguous lineage are derived from hematopoietic stem cells with a potential of multi-differentiation. The differentiation of tumor cells is variable. We need to integrate cell morphology, flow cytometry, pathology, clinical data, and molecular genetics to make a comprehensive diagnosis.

Objective:To explore the feasibility of predicting TP53 mutation risk by immunohistochemical staining (IHC) pattern of P53 in Chinese diffuse large B-cell lymphoma (DLBCL) and its correlation with a prognostic difference.Methods:Between January 2021 and December 2021, 51 DLBCL cases at Beijing Boren Hospital were gathered. These cases had both IHC and next-generation sequencing (NGS) results. IHC classified the P53 protein expression pattern into a loss (<1% ) , diffuse (>80% ) , and heterogeneous (1% -80% ) . The sensitivity and specificity of the predicting TP53 mutation by IHC were assessed by comparing the results of the NGS, and the TP53 high mutation risk group included both loss and diffuse expression of P53. From June 2016 to September 2019, Peking University Cancer Hospital collected 131 DLBCL cases with thorough clinicopathological and follow-up data. From their tumor blocks, tissue microarray blocks were made for IHC evaluation of P53 expression pattern, and prognosis effect of P53 studies.Results:Among 51 cases with both IHC and NGS results, 23 cases were classified as TP53 high mutation risk (7 cases loss and 16 cases diffuse) , 22/23 cases were proved with mutated TP53 by NGS. Only 1 of the 28 cases classified as TP53 low mutation risk was proved with mutated TP53 by NGS. IHC had a sensitivity and specificity of 95.7% and 96.4% for predicting TP53 mutation. NGS identified a total of 26 TP53 mutations with a mutation frequency of 61.57% (13.41% -86.25% ) . In the diffuse group, 16 missense mutations and 2 splice mutations were detected; 6 truncating mutations and 1 splice mutation were detected in the loss group; 1 truncating mutation was detected in the heterogeneous group. Multivariate analysis demonstrated that TP53 cases with high mutation risk have impartial adverse significance for the 131 patients included in survival analysis ( HR=2.612, 95% CI 1.145-5.956, P=0.022) . Conclusion:IHC of P53 exhibiting loss (<1% ) or diffuse (>80% ) pattern indicated TP53 high mutation risk, IHC can predict TP53 mutation with high specificity and sensitivity. TP53 high mutation risk is an independent predictor for adverse survival.

To explore the relationship between progesterone receptor (PGR) gene G1978T polymorphism and endometrial carcinoma. METHODS: After searching PubMed, EMBASE, Wan-fang and CNKI databases for literatures on PGR G1978T genotyping of endometrial cancer patients, the data were extracted and odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using STATA15. The whole blood samples of endometrial carcinoma cases (EC group) and normal women (control group) were collected. Allelic-specific primers matching G1978T wild type G allele and mutant type T allele were designed with 3' terminal phosphorothioate modification, and the two-directional primer extension was performed using Exo + polymerase to genotype PGR gene G1978T polymorphism and Sanger sequencing was used to verify the genotype. RESULTS: PGR gene G1978T mutation was marginally associated with endometrial carcinoma risk (ORper allele =1.10, 95%CI=0.98-1.24, P= 0.072). At the same time, only 1 normal blood samples were found with PGR gene G1978T mutation, and the differences in genotypes and allele frequency between the case group and the control group were not statistically significantP>0.05. CONCLUSION: The G1978T polymorphism of the PGR gene maybe not be associated with the risk of endometrial carcinoma.

Objective: To explore the association between the single nucleotide polymorphism (SNP) of rs175048 in ataxia telangiectasia mutated (ATM) gene and lung cancer susceptibility in Han population. Methods: A total of 225 cases of blood samples from lung cancer patients treated in Hospital of Traditional Chinese Medicine of Hengyang City and the Affiliated First Hospital of Nanhua University from October 2015 to August 2016 were collected as case group, and 128 cases of blood samples from healthy people were collected as the control. The polymorphisms of ATM rs175048 of above mentioned participants were detected by using the SNP sensitive On/Off Switch technique. The genotypes and allele frequencies were analyzed to compare the distribution difference between case group and control group as well as its association to the clinical features of lung cancer. Results: The genotype frequencies of AA, AT and TT of ATM rs175048 were 24.9%, 52.9%, 22.2% in case group and 42.2%, 42.2%, 15.6% in control group, respectively (all P< 0.01). Moreover, the frequencies of alleles A and T were 51.0%, 49.0% in case group, and 63.0%, 37.0% in control group (all P<0.01). Genotype TT might increase while genotype AT might decrease the risk of lung cancer. rs175048 SNP was significantly correlated with smoking, age, sex and family history (all P<0.05). Conclusion: rs175048 SNPis significantly associated with lung cancer, and TT genotype may increase the risk of lung cancer.

OBJECTIVE: To establish a rapid and accurate "on/off" switch technique consisted of 3'-phosphorothioate-modified allele-specific primers and exo+ polymerase to screen the G719S and T790M mutations of epidermal growth factor receptor (EGFR) gene. The switch was used to identify cervical cancer patients who are sensitive to tyrosine kinase inhibitor (TKI). METHODS: Allele-specific primers targeting recombinant wild-type and mutation-type templates were designed with 3' terminal phosphorothioate modification. Two-directional primer extension was carried out using Pfu polymerase. The G719S and T790M mutations were detected by the technique among cervical cancer tissues. The results were verified by Sanger sequencing. RESULTS: No mutation was detected among the 80 cervical cancer cases, and the results were consistent with that of Sanger sequencing. No significant difference was found between the frequencies of the G719S and T790M mutations between the patient and the control groups (P> 0.05). CONCLUSION A sensitive "on/off" switch technique for detecting the two EGFR mutations was established. The G719S and T790M mutations are not associated with cervical cancer.
Carcinoma, Non-Small-Cell Lung ; Drug Resistance, Neoplasm ; ErbB Receptors ; genetics ; Female ; Genes, erbB-1 ; Humans ; Lung Neoplasms ; Mutation ; Protein Kinase Inhibitors ; Uterine Cervical Neoplasms ; genetics