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Objective:To explore the effects of kallikrein 12 (KLK12) on the proliferation, invasion and migration ability of pancreatic cancer cells.Methods:Pancreatic cancer tissues and para-cancer tissues were collected from 95 patients who underwent radical surgical resection, and pathologically diagnosed as pancreatic cancer at the Department of Biliary and Pancreatic Surgery of the First Affiliated Hospital of USTC between February 2014 and October 2018. Expression of KLK12 in pancreatic cancer tissues was investigated using immunohistochemistry, and the correlation between KLK12 expression and clinicopathological characteristics of pancreatic cancer was analyzed. Western blot and qPCR methods were used to detect the expression of KLK12 protein and mRNA in pancreatic cancer cell line SW1990, PANC1 and normal pancreatic gland cells HPDE6-C7. Pancreatic cancer cell lines with up-regulated and down-regulated KLK12 expression were constructed by plasmid transfection. Non-transfected pancreatic cancer cells and transfected pancreatic cancer cells carrying negative control plasmids were used as blank control group and negative control group. CCK8 and Transwell chamber experiments were used to study the changes in cell proliferation, invasion and migration.Results:The positive rate of KLK12 in pancreatic cancer tissues was significantly higher than that in para-cancer tissues (70.5% vs 29.5%, P<0.001), and was significantly related to low tumor differentiation, late TNM stage and lymph node metastasis (all P value <0.05). The expression of KLK12 protein and mRNA in SW1990 and PANC1 cell lines was higher than that in HPDE6-C7 (0.34±0.01, 0.28±0.01 vs 0.21±0.01; 3.31±0.10, 2.91±0.09 vs 1.41±0.20; all P value <0.01). In cells with down-regulated KLK12 expression and then cultured for 72 h, the A450 value of SW1990 cell proliferation (0.94±0.02 vs 1.16±0.05), the number of invading membrane penetrating cells (373.7±14.8 vs 726.0±11.8 per high magnification field) and the number of migrating penetrating cells (696.0±13.1 vs 841.3±15.4 per high magnification field) were significantly decreased. The A450 value of PANC1 cell proliferation (0.96±0.03 vs 1.21±0.03), the number of invading membrane penetrating cells (556.3±13.6 vs 646.0±15.1 per high magnification field) and the number of migrating penetrating cells (449.0±16.5 vs 595.7±8.6 per high magnification field) were also significantly decreased. When the expression of KLKL12 was up-regulated in cells and cultured for 72 h, the A450 value of SW1990 cell proliferation (1.32±0.03 vs 1.11±0.03), the number of invading membrane penetrating cells (556.3±22.2 vs 402.7±10.5 per high magnification field) and the number of migrating penetrating cells (639.3±16.5 vs 433.0±11.8 per high magnification field) were significantly increased. The A450 value of PANC1 cell proliferation (1.26±0.04 vs 1.08±0.03), the number of invading membrane penetrating cells (571.0±17.4 vs 426.7±23.3 per high magnification field) and the number of migrating penetrating cells (740.3±13.0 vs 442.7±10.3 per high magnification field) were also significantly increased (all P value <0.05). Conclusions:KLK12 is highly expressed in pancreatic cancer, and up-regulated KLK12 expression can promote the proliferation, invasion and migration ability of pancreatic cancer cells.
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Objective To explore the effect of tissue kallikrein 1 (KLK1) on mitochondrial function after cardiac ischemia/reperfusion (I/R) injury and its mechanism. Methods After KLK1 overexpression by KLK1 recombinant adenovirus infection, the cardiac I/R rat model was established by ligation of left anterior descending coronary artery and reperfusion. The cardiac infarction area and the apoptosis of cardiomyocytes were detected. The mitochondria were isolated from injured myocardial tissues, and mitochondrial functions (mitochondrial superoxide production, membrane potential and ATP production) determined. The neonatal rat cardiomyocytes were isolated and infected with KLK1 recombinant adenovirus to achieve KLK1 overexpression, and then hypoxia/reoxygenation (H/R) cell model was established. The H/R cells were treated with the media containing bradykinin receptor type 1 (B1R) antagonist R715 or bradykinin receptor type 2 (B2R) antagonist HOE140. The cell viability was determined with MTT assay, and the mitochondrial functions were observed. Results In I/R rats, KLK1 overexpression could alleviate the cardiac injury, and decrease infarction area and cell apoptosis (all P0.01) in cardiac ischemic risk area; moreover, KLK1 overexpression could improve mitochondrial dysfunction, decrease mitochondrial peroxide production, and increase mitochondrial membrane potential and ATP production (all P0.01). In vitro cardiomyocyte model, KLK1 overexpression could also attenuate cardiomyocyte injury (P0.01) and mitochondrial dysfunction (P0.05, P0.01), which could be inhibited by B2R antagonist HOE140. Conclusion KLK1 mitigates mitochondrial dysfunction after cardiac I/R injury, which may be an important mechanism of its cardioprotective effect.
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Objective To study the role of kallikrein 8 (KLK 8) in the pathogenesis of cerebral white matter injury induced by intrauterine infection.Method The pregnant Sprague Dawley rats were randomly assigned into two groups:the observation group and the control group.The rats in the observation group received intraperitoneal injection of lipopolysaccharide (500 μg/kg) by at day 18 and 19 of pregnancy,while the control group received the same dose of saline.The morphology of white matter of the newborn rats were observed at 1 d,3 d,7 d and 14 d after birth.The expression of KLK 8 in the hippocampus was examined using Western blot and reverse transcription-polymerase chain reaction (RTPCR);the concentration of KLK 8 in the serum was measured using enzyme linked immunosorbent assay (ELISA) method at the same time.Result In the observation group,the brain tissue was loose and edematous,the cerebral white matter was weakly stained,and the number of cells reduced.The expressions of KLK 8 in hippocampus in the observation group were higher than the control group [1 d:(0.24 ±0.01) vs.(0.23±0.01),3 d:(0.72±0.02) vs.(0.55±0.04),7 d:(1.08±0.04) vs.(0.84±0.04)],the differences were statistically significant (P ＜ 0.05).The expressions of KLK 8 mRNA in hippocampus of the observation group [1 d:(0.013 ±0.003),3 d:(0.032 ±0.002),7 d:(0.060 ±0.005)] were higher than the control group [1 d:(0.008 ±0.002),3 d:(0.016 ±0.002),7 d:(0.026 ±0.002)],the differences were also statistically significant (P ＜ 0.05).The serum KLK 8 concentration at 1 d,3 d,and 7 d were (5.13 ±0.24) μg/L,(6.46 ±0.24) μg/L,and (7.77 ±0.30) Iμg/L in the observation group,higher than the control group (4.73 ±0.25) μg/L,(5.65 ±0.29) μg/L,and (6.66 ±0.46) μg/L),the differences were also statistically significant (P ＜ 0.05).Conclusion KLK 8 may be involved in the pathogenesis of white matter injury induced by intrauterine infection.
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Pancreatic cancer is a one of the most malignant digestive cancer.Because the lack of effective methods for early diagnosis,most patients have been ineligible for surgical resection when diagnosed.Kallikrein family is a group of serine proteases,because of its ability to decompose the extracellular matrix proteins,it may be closely related to the invasion and metastasis of various cancers.And some members of kallikrein family may become cancer diagnostic biomarkers.This paper reviews all the recent articles about kallikrein family study in pancreatic cancer.
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Objective To investigate the effect of urinary kallidinogenase on serum concentrations of hydrogen sulfide (H2S),neuron-specific enolase (NSE),and S100β in patients with cerebral infarction (CI).Methods From June 2011 to June 2014,80 patients with CI were chosen as study objectives.All patients were divided into two groups:40 patients in study group (urinary kallidinogenase group),and 40 patients in control group.The death rate,the rate of complication and National Institute of Health Stroke Scale (NIHSS) were compared between two groups.The concentrations of H2S,NSE,and S100βwas compared between two groups.Results In study group,the death rate was 5.00％ (2/40),the rate of complication was 22.50％ (9/40);in control group,the death rate was 12.50％ (5/40),the rate of complication was 15.00％ (6/40);and no significant significance was found between two groups (P ＞ 0.05).The NIHSS was (11.2 ± 3.2) in the study group,and (15.7 ± 2.7) in the control group,with statistically significant difference between two groups (P ＜ 0.05).After treatment,the concentrations of H2 S,NSE,and S100β of two groups were decreased significantly (P ＜0.05).At 1w,2w,and 3w,the concentrations of H2S,NSE and S100βhad statistically significant difference between two groups (P ＜ 0.05).Conclusions Urinary kallidinogenase has a cerebral protective effect,which can decrease the concentration of H2S,and increase the concentrations of NSE and S100βin CI patients.
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Objective To study the expression and significance of human tissue kallikrein gene 6(KLK6) in cervical cancer tissues.Methods With glyceraldehyde 3-phosphate dehydrogenase (GAPDH)as reference,the expression of KLK6 in 80 cases of cervical cancer tissues (40 cases with metastasis and 40cases without metastasis) and 40 cases of normal cervical tissues was determined by Taqman probe real-time quantitative reverse transcription-polymerase chain reaction,and analyzed the relationship between cervical cancer occurring and KLK6 expression with clinical data and pathological dats.Results The expression of KLK6 in normal cervical tissues[(1.06 ± 0.40) × 10-3] was lower than that in cervical cancer tissues without and with metastasis[(4.41 ± 1.70) × 10-3,(32.22 ± 6.70) × 10-3],and there was significant difference (P＜0.01).The expression of KLK6 in Ⅰ a,Ⅰ b,Ⅱ a stage of cervical cancer tissues with metastasis was (30.42 ± 5.00) × 10-3,(31.64 ± 1.30) × 10-3,(33.02 ± 8.00) × 10-3,and there was no significant difference among them (P ＞ 0.05).The expression of KLK6 in Ⅰ a,Ⅰ b,Ⅱ a stage of cervical cancer tissues without metastasis was (4.12 ± 1.10) × 10-3,(4.35 ± 1.30) × 10-3,(4.82 ± 1.90) × 10-3,and there was no significant difference among them (P＞0.05).There was significant difference in the expreesion of KLK6 in Ⅰ a,Ⅰ b,Ⅱ a stage between cervical cancer tissues with metastasis and cervical cancer tissues without metastasis (P ＜0.01).Conclusion KLK6 can stimulate the cervical cancer cell proliferation,and participate in the progresses of cervical cancer metastasis.
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Objective To evaluate the expression of human kallikrein10 (KLK10) in different endometrioid tissues and analyse the relationship of KLK10 with ER and PR in endometrioid adenoearcinoma.Methods The expression of KLK10 protein in 12 normal endometria,19 endometrial hyperplasia and 34 endometrial carcinoma were detected by immunohistochemistry.The correlations of the expression of KLK10 protein,ER and PR were analyzed.Results The expression of KLK10 in stage Ⅰ,Ⅱ and Ⅲ were 64.3 ％(9/14),30.0 ％ (3/10),10.0 ％ (1/10),and the difference was statistically significant (P ＜ 0.05).The expression of KLK10 in endometrial carcinoma,normal endometria,endometrial hyperplasia were 66.7 ％ (8/12),33.3 ％(4/12),10.0 ％ (1/10),and the difference was statistically significant (P ＜ 0.05).The expression of KLK10 in G1,G2,G3 were 66.7 ％,33.3 ％,10.0 ％,and the difference was statistically significant (P ＜ 0.05).The positive rate of KLK10 expression was 38.2 ％ and the positive rates of ER and PR expression were 67.6 ％and 55.9 ％,respectively,in 34 endometrial carcinoma.The expression of KLK10 was positively correlated with ER and PR expression (x2 =0.448,P ＜ 0.01).Conclusion Absence or down-regulated expression of KLK10 may play an important role in the formation and development of endometrioid adenocarcinoma.The low expression of KLK10 is correlated with low expression of ER and PR in endometrioid adenocarcinoma.The positive expression of KLK00,ER and PR predicts a better prognosis.
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Objective To investigate the effects of tissue kallikrein on expressions of bradykinin, bradykinin Bl receptor (B1R) and bradykinin B2 receptor (B2R) in ischemic brain tissue following cerebral ischemia-reperfusion in rats. Methods Fifty-four SD rats were randomly divided into three groups: sham operation, normal saline (NS) (2 ml · kg-1 · d-1, for 3 days), and TK (IK 175 × 10-3 U· kg-1 · d-1,for3 days) groups (n = 18 in each group). After three days,the neurological deficit score and the measurement of cerebral infarct volume were performed,The concentration of bradykinin in the ischemic region was detected by the enzyme- linked imrnunosorbent assay (ELBA).reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the mRNA and protein expressions of BlR, B2R in ischemic brain tissue, respectively. Results Compared with the NS group, the neurological deficit (6.17 ± 1. 17 vs. 8. 17 ± 1.33; t =2.000, P =0- 004) and the cerebral infarct volume (29. 67% ±3. 78% vs. 37. 50% ± 6. 72% ;t =0.078, P =0.005) in the TK group were reduced significantly; the concentration of bradykinin in ischemic brain tissue in the TK group was increased significantly (9.25 ± 1. 13 vs. 15.53 ± 1.68, t =6.283, P =0. 000); the expression of B2R mRNA was up regulated significantly (1. 21 ±0. 17 υs. 2.15 ±0.20; t =0.943,P =0- 000), but the up-regulation of the B2R mRNA expression was not obvious (0.51 ±0.05 υs. 0.57 ±0.06; t =0.058, P =0. 141); the expression of B2R protein in ischemic brain tissue was up-regulated significantly (1. 15 ±0. 16 vs. 1. 88 ± 0.21, t =0. 737, P =0. 000), but the up-regulation of BlR was not obvious (0. 50 ±0.04 vs. 0.53 ±0.05, t = 1.326, P =0. 214). Conclusions TK has protective effect on cerebral ischemia-reperfusion in rats. It may increase the bradykinin concentration in ischemic brain tissue, and up-regulate B2R expression, but it has little effect on Bl R expression.It is speculated that B2R may play a major role in TK protecting ischemic brain tissue.
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Objective To investigate the interference and associated mechanism of hnman tissue kallikrein (HK) gene on renal interstitial fibrosis in rats with 5/6 nephrectomy. Methods Human kallikrein cDNA was packed in a recombinant adeno-associated virus(rAAV)-based plasmid vector. The rAAV-HK was produced by transfection in 293 cells. Twenty-four male Wistsr rats were divided into sham operation and operation groups. The rats with 5/6 nephrectomy were randomly divided into simple operation, control and experiment groups. The rats in experiment group received single dose rAAV-HK via the tail vein with 1×1011 pfu. Before nephrectomy and every month after surgery until the rats were sacrificed, the caudal arterial pressure was measured using tail cuff blood pressure determinator. Three months after HK gene delivery, the rats were sacrificed. The expression of HK in rats was assessed by RT-PCR, Western blot and enzyme-linked immunosorbent assay (ELISA). The pathological changes of renal interstitium were evaluated by Masson stainning, and the distribution of bradykinin B2 receptor (BKB2R) and angiotensin Ⅱ typel receptor (ATIR) was examined by immunohistochemistry. The expressions of BKB2R, AT1R, p-MAPK protein in renal tissue were detected by Western blot. Results Three months after HK gene delivery, the systolic blood pressure of experiment group was significantly decreased compared with the control group [(163±13) nun Hg vs (217±16) mm Hg, P<0.01](1 mm Hg=0.133 kPa). Compared with sham rats, the rats in simple operation group and control group had much more renal interstitial collagen deposition and more serious fibrosis performance, but renal interstitial collagen deposition and fibrosis were significantly ameliorated in the rats of experiment group. In addition, the tubulointerstitial injury index of HK transgenic rats was significantly lower than that of the rats in control group (1.33±0.73 vs 3.01±0.62, P<0.01). Up-regnlating expression of bradykinn B2 receptor protein and down-regulating expression of AT1 receptor and p-MAPK protein were found in renal tissues of experimental group after three months (P<0.05). Conclusion HK gene delivery significantly alleviates renal interstitial fibrosis in rats with 5/6 nephrectomy through regulating the expression of bradykinin B2 receptor, AT1 receptor and p-MAPK in renal tissue.
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AIM: To study the effect of tissue kallikrein gene (HK) treatment on blood pressure in type 2 diabetic rats and its mechanism. METHODS: Male Wistar rats were injected with low dose streptozotocin and fed with diets enriched in fat and sugar to form type 2 diabetic model. Recombinant adeno-associated viral vectors (rAAV)-mediated HK gene (HK group) or LacZ gene (LacZ group) was introduced to the diabetic rats. The systolic blood pressure was measured every 2 weeks. The acetylcholine (Ach)-dependent vasodilation response, the synthesis of nitric oxide (NO), the expression of endothelin-1 (ET-1) and endothelin-A receptor (ET_A-R) in the aorta were detected. RESULTS: (1) Systolic blood pressure was significantly higher in diabetic rats than that in normal control rats. In HK group, systolic blood pressure was significantly reduced within 2 weeks after injection with rAAV?HK, reached near normal levels at 4 weeks and kept until the experiments ended (16 weeks). (2) In LacZ group, Ach-dependent vasodilation response of isolated aorta was markedly decreased than that in HK group (P