Objective To establish the methods of isolating and culturing human ovarian endometriosis-derived microvascular endothelial cells (OEMEC).Methods The tissues of human endometriotic cyst of ovary were finely minced with scissors,then digested by collagenase Ⅰ,Ⅱ and trypsinethylene diamine tetraacetic acid (EDTA).The cells were purified by using centrifugation of 2000 r/min speed.OEMEC were identified by light microscope and transmission electron microscope observing CD34,FⅧ-Rag and Weibel-Palade in microvascular endothelial cells.Results The OEMEC grew as confluent monolayer like cobblestones under light microscope.CD34 and FⅧ-Rag were expressed strongly,and the percentages of CD34 and FⅧ-Rag positive cells were 91.4％ and 92.5％.Weibel-Palade bodies could be observed under transmission electron microscope.The time of cell doubling proliferation was 4.5 days.Conclusion The established system of isolating OEMEC would provide lab base for studying the mechanisms of angiogenesis in endometriosis lesions.

AIM:To detect the treatment of K562 leukemia cells with bortezomib altering the expression of genes fas,bcl-2,bcl2l12,bim,bax,caspase-9 and caspase-3.METHODS:MTT assay was used to detect the inhibition of proliferation.Apoptosis was detected by Annexin-V staining and mitochondrial transmembrane potential(??m).RT-PCR was used to analyze the mRNA expressions of fas,bcl-2,bcl2l12,bim,bax,caspase-3 and caspase-9.RESULTS:Bortezomib caused a time-and dose-dependent inhibition of cell proliferation and IC50 of 24 h and 48 h were 161.41 nmol/L and 96.33 nmol/L,respectively.At the concentration of 104 nmol/L,bortezomib induced apoptosis in a time-dependent manner,including increasing annexin-V positivity and decreasing the ??m.RT-PCR showed that bortezomib up-regulated the mRNA expression of fas,bcl2l12,caspase-9 and caspase-3,but mRNA expressions of bcl-2,bim and bax did not changed obviously.CONCLUSION:Bortezomib inhibits the proliferation of K562 and induces apoptosis,in which fas,bcl2l12,caspase-9 or caspase-3 gene is one of the main genes taking part in.

Objective To evaluate the growth inhibition of human gastric carcinoma cell lines SGC 7901 in vitro and the expression of bcl-2, bcl 2l12 and bax with docosahexaenoic acid (DHA) and 5-fluorouracil (5-FU). Methods The effect of DHA and 5-FU was measured by trypan blue, and the interaction between two agents was judged by combination index (CI). Cells were observed by inverted microscope. Flow cytometry was used for analysis of apoptosis by PI staining and Annexin-V/PI. RT-PCR was used to analyze the levels of bcl-2, bcl 2l12 and bax mRNA. Results DHA significantly inhibited the growth of SGC 7901 cells in a dose- and time-dependent way ( P < 0. 05 ), the IC50 of 24 h and 48 h was 67. 81 μg/ml and 45.76 μg/ml, and a strong synergism was found in the combination of DHA and 5-FU (CI < 1 ,P <0. 01 ). Treated by DHA and 5-FU for 48 h, cells became sparse under inverted microscope. DHA or 5-FU was able to induce apoptosis and the effect became even more significant by the combination of DHA and 5-FU. Cells were holted in phase of G01/G1 and S. RT-PCR showed that DHA or 5-FU down-regulated the expression of bcl-2 and bcl 2l12 mRNA, while bax mRNA expression was not downregnlated. Conclusions DHA could inhibit the growth of gastric carcinoma cells, DHA and 5-FU had synergetic effect in the inhibition of the cells growth and blockage of the cell cycles possibly by down-regulating the expression of bcl-2 and bcl 2l12.

Objective:To observe the effect of moxibustion on T lymphocyte subsets in peripheral blood of rats with gastric cancer. Methods:Sixty healthy Sprague-Dawley (SD) rats were adaptively fed for one week. By the random number table method, 10 rats were randomly selected as a blank group, and 12 rats were randomly selected to simulate the tumor transplantation process; after transplantation, 10 rats were randomly selected as a sham operation group. The remaining 38 rats were used to prepare gastric cancer models by gastric transplantation of the Walker-256 tumor tissue; 8 rats were randomly selected to verify the successful modeling after 7 d; the remaining 30 rats were randomly divided into a model group, a moxibustion group and an infrared group by the random number table method, with 10 rats in each group. From the first day of enrollment, the rats in the moxibustion group received mild moxibustion at Zhongwan (CV 12), Guanyuan (CV 4) and bilateral Zusanli (ST 36) (the first group) and bilateral Pishu (BL 20) and Weishu (BL 21) (the second group), and the two groups of acupoints were alternated every other day. The rats in the infrared group received infrared radiation on the stomach area and the area on the back between the T12-T13 spinous processes, the two areas were alternated every other day. Rats in the moxibustion group and the infrared group were treated for 20 min each time, once a day for 21 d. Rats in the blank group, the sham operation group, and the model group were simultaneously grasped and fixed, and no other treatment was performed. After 21 d of intervention, the rats in each group were fasted for 12 h, and blood was collected from the orbits. The numbers of CD3+, CD3+CD4+, CD3+CD8+ T lymphocytes in peripheral blood were determined by flow cytometry, and the ratio of CD3+CD4+/CD3+CD8+ was calculated. The rats were sacrificed and the thymus was dissected under sterile conditions to calculate the thymus index. Results:Compared with the blank group, the thymus index, peripheral blood CD3+, CD3+CD4+, CD3+CD8+ and CD3+CD4+/CD3+CD8+ ratio in the sham operation group did not change significantly (allP>0.05). Compared with the blank group, the thymus index of the model group was increased (P<0.05), the CD3+ and CD3+CD8+ T lymphocytes were increased (bothP<0.01), and the CD3+CD4+/CD3+CD8+ ratio was decreased (P<0.05). Compared with the model group, the thymus index of the moxibustion group was increased (P<0.01), and CD3+, CD3+CD4+ and CD3+CD8+ T lymphocytes and the ratio of CD3+CD4+/CD3+CD8+ in peripheral blood were increased (allP<0.05). Compared with the infrared group, the thymus index of the moxibustion group was significantly increased (P<0.05), the CD3+ and CD3+CD4+ T lymphocytes in the peripheral blood were significantly increased (bothP<0.01), and the CD3+CD8+ was increased (P<0.05). Conclusion:Moxibustion can significantly increase the thymus index of gastric cancer-bearing rats and activate CD3+CD4+ and CD3+CD8+ T lymphocytes in peripheral blood.

Objective:To observe the effect of moxibustion on the mRNA and protein expressions of heat-shock protein 70 (HSP70) in gastric cancer-bearing rats. Methods: A total of 40 healthy Sprague-Dawley (SD) rats were adaptively fed for one week. The gastric cancer model was prepared by Walker-256 cancer tissue transplantation. After 7 d, 10 rats were randomly selected to verify the successful modeling, and the remaining 30 rats were divided into a model group, a moxibustion group and an infrared group by the random number table method, with 10 rats in each group. After enrollment, the moxibustion group received suspended moxibustion at Zhongwan (CV 12), Guanyuan (CV 4) and bilateral Zusanli (ST 36), (the first group of acupoints) on the 1st day, and suspended moxibustion at bilateral Pishu (BL 20) and Weishu (BL 21), (the second group of acupoints) on the 2nd day, 20 min each time, once a day. Moxibustion was alternately performed every other day at the two groups of acupoints for 21 d. From the day of enrollment, rats in the infrared group were irradiated with the infrared radiation at the stomach area on the 1st day, and at the T12-T13 interspinous region on the 2nd day, 20 min each time, once a day, and the two locations were alternately irradiated every other day for 21 d. During the treatment, rats in the model group were intervened by grasping and fixation without treatment. At the end of the treatment, blood was collected from the inner eye orbit, and the HSP70 expression in peripheral blood was determined by enzyme linked immunosorbent assay (ELISA). Rats were sacrificed, the tumor volume and growth inhibition rate were measured. The position and changes of HSP70 in gastric cancer were observed by streptavidin-perosidase (SP); HSP70 protein expression was determined by ELISA; HSP70 mRNA expression in cancer tissues was determined by reverse transcription-polymerase chain reaction (RT-PCR) assay. Results: In comparison of the model group, the volume growth of the gastric cancer in the moxibustion group was significantly restricted (P＜0.01); the volume growth inhibition rate in the moxibustion group was 37.93%; the HSP70 expression in peripheral blood and the cancer tissues was significantly increased (both P＜0.01); the expression of HSP70 mRNA and HSP70 content in gastric tumor were both obviously increased in the moxibustion group (P＜0.01); and a large amount of HSP70 was released to the outside of cancer cells in the moxibustion group. In comparison of the model group, the volume growth of the gastric cancer in the infrared group was slightly restricted (P＜0.05) with a volume growth inhibition rate of 15.89%; the HSP70 expression in the infrared group was increased significantly in peripheral blood (P＜0.01) and in the gastric cancer tissues (P＜0.05); more HSP70 was released outside of the cancer cells in the infrared group. In comparison of the infrared group, the volume growth of gastric cancer was more restricted in the moxibustion group (P＜0.05), and the HSP70 expression in the gastric cancer tissues was also higher (P＜0.05); more HSP70 was released outside of the cancer cells in the moxibustion group. Conclusion: Moxibustion and infrared treatment inhibit the gastric cancer growth in the gastric cancer-bearing rats, up-regulate the HSP70 expression in gastric cancer tissues, and promote the production and extracellular release of HSP70, and the effect of moxibustion is more obvious.

Objective To analyze the clinical characteristics of Duchenne/ Becker muscular dystrophy (DMD/BMD)with the initial presentation of transaminase elevation,in order to improve the clinician's understanding of this disease,and reduce misdiagnosis and missed diagnosis. To investigate the relationship between the elevation of transami-nase and the early stage of DMD/ BMD,and to provide the strategy for early diagnosis. Methods Twenty - four pa-tients admitted to the hospital with elevated serum transaminase as the initial presentation from January 2012 to Decem-ber 2014,who were diagnosed as DMD/ BMD by genetic testing or muscle biopsy,were enrolled. Their clinical data and laboratory examinations were retrospectively analyzed,including clinical features,diagnostic steps,serum muscle en-zymes,genetic analysis,electromyography and muscle pathological changes. Results The 24 patients were all male without family history of DMD/ BMD prior to birth. The average visiting age was (3. 4 ± 1. 2)years (ranging from 0. 8 to 6. 1 years),and 87. 5％ (21 / 24 cases)of cases were preschool children aged 2 - 6 years. Hypertransaminasemia was found in 21 cases (87. 5％)during the kindergarten physical examination,1 case during pre - operative investigation and 2 cases during respiratory infection. Due to its insidious onset,the time interval between incidental finding of elevated transaminase and definitive diagnosis was between 0. 6 and 20. 4 months. Among them,16 cases (66. 7％)had obvious pseudohypertrophy of calf muscles,and 18 cases (81. 8％)showed different degrees of movement disorder,such as unable to jump,easy to fall,and difficulty in climbing stairs. In addition,18. 2％ cases (4 / 22 cases)had a delay in language development. The serum alanine aminotransferase and aspartate aminotransferase levels were 120. 3 - 761. 7 U/ L and 83. 3 - 675. 5 U/ L,respectively. Serum creatine kinase (CK)was found to be markedly elevated (ranging from 3940 to 27510 U/ L)in all patients. Electromyography showed myogenic damage in 13 / 23 cases (56. 5％). DMD gene deletions were found in 18 cases (75. 0％),and duplications in 4 cases (16. 7％). The muscle biopsy performed in 2 cases (8. 3％)multiplex ligation - dependent probe amplification (MLPA)- negative cases showed evidence of dystrophic features on routine histology. Immunohistochemistry showed absent dystrophin staining in all 2 MLPA - nega-tive cases. Conclusion The clinical manifestation of DMD/ BMD is not typical in the early stage,so it is easy to be neglected or misdiagnosed. Elevated ALT and AST are important clues in the early diagnosis of DMD/ BMD. Children with elevated transaminase,in the absence of other signs and symptoms of hepatic injury,may have occult muscular di-sease,most frequently DMD/ BMD. A thorough physical examination and history taking as well as the measurement of serum CK are helpful in the differential diagnosis. Genetic testing or muscle biopsy should be done early for correct diagnosis of DMD/ BMD.

Objective To evaluate the diagnostic value of rectal endoscopic ultrasonography for recto-vaginal endometriosis (RVEM).Methods The clinical data of 36 patients who met the criteria for RVEM between September 2009 and September 2016 in Shengjing Hospital were analyzed.Rectal endoscopic ultrasonography was compared with colonoscopy and pelvic magnetic resonance imaging (MRI) in terms of preoperative diagnosis and their conformance with surgical and postoperative pathological findings.Results Rectal endoscopic ultrasonography was superior to colonoscopy and pelvic MRI in diagnosing RVEM,invasive bowel disease,and the invasive level in the preoperative assessment (P ＜ 0.05),and in its conformance with surgical and postoperative pathological findings.Conclusion Rectal endoscopic ulwasonography is a reliable technique for diagnosing RVEM,with good accuracy in the preoperative diagnosis of invasive bowel disease and its level of invasiveness.

Objective To investigate the correlation of body mass index (BMI) on pathological complete response (pCR) and prognosis of breast cancer patients receiving neoadjuvant chemotherapy.Methods A retrospective analysis was performed on 98 cases of breast cancer patients in the Tumor Hospital Affiliated to Zhengzhou University from December 2013 to November 2015.Patients were divided into normal or underweight (N/U,BMI ＜25.0 kg/m2) group,overweight (OW,BMI 25.0-29.9 kg/m2)group and obese (OB,BMI ≥30.0 kg/m2) group according to BMI.A total number of 50 (51.0％) patients were N/U,32 (32.6％) patients were OW,and 16 (16.3％) patients were OB.The pathological complete remission after neoadjuvant chemotherapy was observed,and the prognostic evaluation index was disease-free survival rates.Univariate analysis of BMI and pCR correlations was performed by Chi-square test or Fisher exact test,and logistic regression for multivariate analysis.Kaplan-Meier analysis and log-rank test were used to analyze survival status,and Cox proportional hazard model analysis for multivariate analysis.Results In this study,A statistically significant difference was found in the molecular subtypes of the three groups(P ＜ 0.05).Twenty-nine(29.6％) patients achieved pCR,pCR rates in N/U group,OW group and OB group were 36.0％ (18/50),25.0％ (8/32),and 18.8％ (3/16),respectively,but the difference was not statistically significant (P ＞ 0.05).Multivariate logistic regression analysis showed that obesity and molecular subtype were independent factors of pCR in all patients (P ＜ 0.05).The 3-year disease free survival rates of the N/U,OW,and OB groups were 84.0％,93.5％ and 80.4％,respectively (P ＞ 0.05).Multivariate survival analysis showed that the BMI was not an independent prognostic factor for the 3-year disease free survival rate (P ＞ 0.05).Conclusion Excessive BMI (obesity) is an independent predictor of pCR in breast cancer patients receiving neoadjuvant chemotherapy,but does not affect the prognosis of these patients.

Objective:To explore the inhibitory effect of moxibustion on tumor growth and metastasis, and also its possible mechanism, in gastric tumor-bearing rats by investigating the expressions of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF). Methods:Fifty healthy Sprague-Dawley (SD) rats (half male and half female) were routinely housed for 1 week. A total of 20 rats were randomly divided into a blank group and a sham operation group, with 10 rats in each group. The remaining 30 rats were used to make gastric cancer models by implantation of ascites-type Walker-256 cancer cells. After successful modeling, rats were randomly divided into a model group, a moxibustion group and an infrared group, with 10 rats in each group. From the day of modeling, the body weight of each group was weighed every 4 days. Warm moxibustion was alternately performed at two-group acupoints [Zhongwan (CV 12), Guanyuan (CV 4) and bilateral Zusanli (ST 36) in one group, and bilateral Pishu (BL 20) and Weishu (BL 21) in another group] in the moxibustion group. The body surface projection area of the stomach was irradiated with short-wave infrared rays in the infrared group, once a day, 20 min per time for 21 d. At the end of the treatment, the gastric tumor was completely dissected, and the tumor volume and tumor growth inhibition rate were calculated. Then the gastric tumor cell metastasis was recorded. The levels of VEGF and EGF in rat gastric tumor tissues were determined by enzyme-linked immunosorbent assay (ELISA). Results:Compared with the blank group, the body weight of the model group decreased significantly after modeling (P<0.05); compared with the model group, the rats in the moxibustion group had increased body weight during the middle and late stages (bothP<0.05). The tumor volumes of rats in the moxibustion group and the infrared group were smaller than the volume in the model group (bothP<0.05). The tumor growth inhibition rate in the moxibustion group was significantly higher than that in the infrared group (P<0.05). The case number of tumor metastasis in the moxibustion group was smaller than that in the model group and the infrared group. The VEGF level in the tumor tissues of the model group was statistically significantly higher than that in the blank group (P<0.05). Compared with the model group, the VEGF levels in the moxibustion group and the infrared group were statistically significantly lower (bothP<0.05). The EGF levels in the tumor tissues of the model group was statistically significantly lower than that in the blank group (P<0.05); compared with the model group, the EGF levels in the moxibustion group and the infrared group were statistically significantly increased (bothP<0.05). Conclusion:Moxibustion can increase the body weight, inhibit the tumor growth, invasion and metastasis in gastric tumor-bearing rats, which may be related to the regulation of VEGF and EGF expressions in tumor tissues.

Abstract： Objective　To analyze the value and influencing factors of cross-primer isothermal amplification technology(CPA) in clinical screening and diagnosis of tuberculosis (TB). Methods　We collected 543 inpatients in the Second Affiliated Hospital of Hainan Medical College from January 1, 2018 to December 31, 2021, including 179 patients with tuberculosis, 187 patients with pneumonia and 177 patients with other diseases. The patients' sputum, alveolar lavage fluid, pleural effusion and midstream urine were detected by CPA, smear microscopy, culture method and gene detection. The value of CPA detection in the diagnosis of tuberculosis and its influencing factors were evaluated. Statistical analysis was performed using SPSS 26.0. Results　The total positive rate of CPA was 14.4% (78/543), and the positive rate of sputum samples accounted for 29.1% (39/134). Among the 78 cases of CPA positive patients, the tuberculosis group accounted for 69.2% (54/78), followed by pneumonia group 21.8% (17/78), and other diseases group accounted for 9.0% (7/78). Taking CPA test as the reference method, the "sensitivity" of smear microscopy was lower than that of genetic testing and culture, while the "specificity" was higher than that of culture and gene testing, and the "missed diagnosis rate" of smear microscopy was higher than that of genetic testing and culture. CPA test positive was related to gender, ESR and pneumonia. There is a good agreement between CPA test and culture method and gene test (Kappa>0.9), and a moderate agreement between CPA test and smear microscopy (Kappa=0.616). Conclusions　Sputum specimen is the best choice for CPA detection, while the value of pleural effusion detection is relatively limited. Sputum, alveolar lavage fluid and midcourse urine can be used as clinical specimens for screening and diagnosis of "tuberculosis group and other disease group", while sputum can be used for screening and diagnosis of "tuberculosis group and pneumonia group". Gender, ESR and pneumonia are the influencing factors of CPA positive patients. Therefore, CPA testing is worthy of clinical promotion, but more clinical research data are needed.