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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 investigate the effects of human urinary kallidinogenase (HUK) on cerebral perfusion,inflammatory marker level,neurological deficits,and short-term clinical outcomes in patients with acute ischemic stroke who exceeded the time window of thrombolytic therapy.Methods Patients with acute ischemic stroke exceeded the time window of thrombolytic therapy and treated in the Department of Neurology,the Second Affiliated Hospital of Nantong University from June 2016 to March 2018,and performed magnetic resonance perfusion imaging on the 1st and 14th d after admission were enrolled retrospectively.Patients were divided into HUK and control groups according to whether they received HUK treatment or not.All patients underwent magnetic resonance perfusion imaging and serum inflammatory markers measurement on the 1st and 14th d after admission.The National Institute of Health Stroke Scale (NIHSS) scores,cerebral perfusion levels,and serum inflammatory marker levels were compared between the 2 groups.On the 14thd after admission,the outcome was determined according to the modifiel Rankin Scale (mRS) scores.0-2 was defined as good outcome and ＞2 was defined as poor outcome.Multivariate logistic regression analysis was used to determine the independent influencing factors of outcome.Results A total of 62 patients with acute ischemic stroke exceeded the time window of thrombolytic therapy were enrolled,including 37 patients in the HUK group and 25 in the control group.There were no significant differences in the demographic and other baseline data between the HUK group and the control group,except for baseline NIHSS score (P =0.049).The reduction of NIHSS score after treatment in the HUK group was more significant than that in the control group (P ＜0.01).Serum high-sensitivity C-reactive protein level in the HUK group decreased significantly after treatment (P ＜ 0.01),and plasma lipoprotein-associated phospholipase A2 also showed a downward trend.In terms of perfusion imaging parameters,the relative cerebral blood flow was significantly increased after treatment in the HUK group,and the relative mean transit time and relative peak time were significantly decreased.The above increase and decrease were statistically significant compared with the control group (all P＜0.05).On the 14th d after admission,the mRS score showed that 51 patients had a good outcome and 11 had a poor outcome.Multivariate logistic regression analysis showed baseline NIHSS scores (odd ratio [OR] 2.545,95％ confilence interval [CI]1.124-5.541;P=0.024),atrial fibrillation (OR 5.712,95％ CI 1.737-24.685;P=0.039),and cardiogenic embolism (OR 4.485,95％ CI 1.148-18.262;P =0.040) were the independent risk factors for poor outcomes,and whether using HUK was not significantly associated with the outcomes.Conclusion For patients with acute ischemic stroke that exceeds the time window for thrombolysis,HUK improves cerebral perfusion,reduces inflammation,and improves neurological deficits in patients,but does not improve short-term neurological outcomes.
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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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Objective To investigate the effect of human urinary kallidinogenase (HUK) on cerebral ischemia reperfusion injury in mice.Methods One hundred and ten male ICR mice were randomly divided into sham operation,control and HUK groups.A cerebral ischemia-reperfusion model was induced by transient middle cerebral artery occlusion.The infarct volume was detected by triphenyltetrazolium chloride staining.Bcl-2,Bax,and caspase-3 expression levels in the ischemic cortex were detected by Western blot.Bcl-2 and Bax positive cells in the hippocampal CA1 area on the ischemic side were detected using Immunohistochemical staining.Apoptotic cells in the ischemic cortex were detected by TUNEL staining.Results No infarction and neurological deficits were found in the sham operation group.At 24 h after ischemia-reperfusion,the infarction voltne (P ＜0.01) and neurological deficit score (P =0.02) in the HUK group were significantly lower than those in the control group;at 72 h after ischemia-reperfusion,the infarction volume (P ＜ 0.01) and neurologic deficit score (P =0.03) in the HUK group were also significantly lower than those in the control group.Westem blot analysis showed that the expression level of Bcl-2 in the ischemic cortex in the HUK group was significantly higher than that in the control group (P ＜ 0.001),and the expression levels of caspase-3 (P ＜ 0.001) and Bax (P ＜ 0.001) in the cerebral cortex in the HUK group were significantly lower than those in the control group.No apoptotic cells were found in the sham operation group.The number of apoptotic cells in hippocampal CA1 area (P ＜ 0.01) and the number of Bax positive cells (P ＜0.01) in the HUK group were significantly less than those in the control group,while the number of Bcl-2 positive cells was significantly more in the control group (P ＜ 0.01).Conclusions HUK has a certain protective effect on ischemia-reperfusion injury in mice,its mechanism may be associated with the upregulation of Bcl-2 protein expression and downregulation of caspase-3 and Bax protein expression,thus inhibiting cell apoptosis.
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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 evaluate the influence of ultrasound-mediated contrast agent microbubbles carrying kallidinogenase targeted therapy on neurogenesis and angiogenesis after experimental acute cerebral infarction.Methods Kallidinogenase-loaded microbubbles were prepared using mechanical shaking method.Middle cerebral artery occlusion (MCAO) model was established in male Wistar rats by sutureoccluded method.MCAO rats (n =120) were randomly divided into 5 groups:ultrasound-mediated kallidinogenase-loaded microbubbles group (group 1),ultrasound-mediated microbubbles group (group 2),ultrasound-mediated saline group (group 3),kallidinogenase group (group 4),saline group (group 5).Medication was given through tail vein daily for 2-6 consecutive days starting 24 h after MCAO.Ultrasound groups (groups 1,2 and 3) were given 2 MHz pulse ultrasonic irradiation on the ischemia lateral skull for 10 min after injection.Cell proliferation was examined using 5'-bromo-2'-deoxyuridine (BrdU,50 mg/kg).Infarction volume and neurological function were evaluated on the 3rd,7th day after MCAO respectively.Doublecortin (DCX) + cells in the subventricular zone and laminin + in the peri-infarction region were observed at the same time.Results The number of DCX + cells in the subventricular zone (under 20 × ocular) of groups 1-5 was 251.8 ± 13.1,125.7 ± 11.6,130.2 ± 13.7,234.5 ± 12.4 and 123.7 ± 10.0 respectively.The percentage of laminin + cells in the peri-infarction region (× 10 objective) of groups 1-5 was 10.0％ ± 0.8％,5.2％ ± 0.7％,5.0％ ± 1.0％,8.0％ ± 1.8％ and 5.0％ ± 0.9％ respectively.The number of DCX + cells in the subventricular zone and laminin + cells in the peri-infarction region of the group 1 and the group 4 was significantly more on the 7th day after MCAO compared with those of the other three groups (DCX:t values of group 1 vs groups 2,3,5 were 17.88,17.17 and 18.16,all P ＜ 0.01 ; t values of group 4 vs groups 2,3,5 were 15.42,14.78 and 15.70,all P ＜0.01.Laminin:t values of group 1 vs groups 2,3,5 were 7.01,6.71 and 7.11,all P ＜ 0.01 ; t values of group 4 vs groups 2,3,5 were 4.23,3.94 and 4.33,all P ＜0.01).Moreover,the number of DCX+ cells in the subventricular zone (t =2.46,P ＜ 0.05) and laminin + cells in the peri-infarction region (t =2.78,P ＜ 0.05) of the group 1 was much more than those of the group 4.Neurologic scores on the 7th day of groups 1-5 were 1.00 (0.75,1.25),2.0 (2.00,3.00),2.0 (1.00,2.00),1.5 (0.75,2.00),2.0 (2.00,2.50).Compared with other four groups,group 1 showed better functional improvement after stroke (U values of group 1 vs groups 2-5 were 2.0,4.0,7.5 and 2.5,all P ＜ 0.05).While there was no significant difference in infarction volume among five groups at all the time points after MCAO.Conclusions Our study demonstrates ultrasound-mediated kallidinogenase-loaded contrast agent microbubbles targeted therapy promotes neuroblasts proliferation and vascular regeneration compared with mediated and non-medicine-loaded microbubbles therapy,which attributes to functional improvement after MCAO.Therefore,ultrasound-mediated ultrasound contrast agent microbubbles carrying drugs targeted therapy may have a perspective on ischemic stroke.
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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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BACKGROUND:Exercise can elicit generation of coronary col ateral circulation through numerous pro-angiogenic growth factors. It is not enough via a single factor to clearly definite signaling pathways for the generation of col ateral circulation. Many pro-angiogenic growth factors are involved in kal ikrein-kinin system, and there is no report about exercise effects on this system. OBJECTIVE:To study effects of exercise training on bradykinin expression in rats with myocardial infarction. METHODS:Thirty Wistar rats were randomly assigned to control group, myocardial infarction group and exercises training group. Rats in the control group were subjected to thoracotomy with no coronary artery ligation. Rats in the other two groups were modeled. In the exercise training group, 3 days after myocardial infarction, rats were subjected to 30-minute exercise training on treadmil , once a day for 4 weeks. At the terminal of the experiment, blood samples were obtained to analyze bradykinin expression by enzyme linked immunosorbent assay, and the myocardium was sampled to analyze relative blood flow by microsphere method. RESULTS AND CONCLUSION:After exercise training, bradykinin level in the exercise training group was significantly higher than that in the myocardial infarction group (P<0.001). At the end of experiment, the relative blood flows of the exercise training and myocardial infarction groups were both increased compared with before experiment (P<0.05, P<0.001). At the end of experiment, the relative blood flow of the exercise training group was significantly higher than that of the myocardial infarction group (P<0.01). In al the three groups, bradykinin level was significantly correlated with the relative blood flow in the myocardium. These findings indicate exercises training can improve bradykinin expression apparently and increase blood flow in the myocardium, which il ustrates that the kal ikrein-kinin system plays an important role in exercise induced angiogenesis.
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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 study the effects of urinary kallidinogenase (kallikrein) on focal cerebral blood flow (CBF) following cerebral infarction in rats by laser speckle imaging.Methods Permanent middle cerebral artery occlusion (MCAO) was induced in male Sprague-Dawley rats by the intraluminal filament technique.Laser speckle imaging was used to measure CBF in the ischemic cortical area and middle cerebral artery territory.The brain was stained with 2,3,5-triphenyltetrazolium chloride (TTC) to determine the infarct size.Neurological deficit score was measured.Results CBF increased in both hemispheric cortical area and MCA territory on the first and second days following urinary kallikrein administration at high dose but not at low dose.Larger blood vessel diameter and increased blood flow velocity were noticed in the high dose group in some arteries when compared to the low dose group and normal saline control group.At 36 h after cerebral ischemia,the brain infarct size was 10.14% ±3.02% ,25.99% ±3.90% and 27.10% ±3.32% in high, low dose and normal saline control groups,respectively.The infarct size was significantly smaller in the high ( F = 61.14, P<0.01 ) but not low dose group when compared to the normal saline control group.The neurological deficit was milder in the high dose group but not the other two groups at 4 h after cerebral ischemia; however, there were no differences among the groups at 36 h after MCAO.Conclusions Urinary kallidinogenase can reduce cerebral infarction volume and neurological deficit in rats following focal cerebral ischemia.These effects may be attributed to enhanced collateral circulation and improved CBF in the hemispheric cortical area and MCA territory.
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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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Objective The aim of this study was to investigate the clinical value of human serum Kallikrein 6 for the diagnosis and monitor of pithelial ovarian cancer. Methods Serum levels of KLK6 were analyzed with ELISA in 30 cases of epithelial ovarian carcinoma, 20 cases of benign ovarian tumor and 30 cases of healthy women. In the meantime, serum CAi25 was determined with chemiluminescence. Furthermore, serum levels of KLK6 and CA125 were also detected in 12 case of epithelial ovarian carcinoma with the same methods one week and the 3rd month postoperation of follow-up. Results Serum levels of KLK6 in epithelial ovarian carcinoma was higher than that in benign ovarian tumor and healthy women (P < 0.05). KLK6 also showed positive correlation with clinical stage, cytological grade, pelvic lymph node metastasis, recurrent or dead disease (P < 0. 05). On the contrary, KLK6 showed no significant correlation with pathological types (P >0. 05). After surgery of follow-up, KLK6 and CA125 were significantly decreased in 12 case of epithelial ovarian carcinoma (P < 0. 05). Furthermore, the total sensitivity and specificity of KLK6 in the diagnosis of epithelial ovarian carcinoma was 73.3% and 85.0% respectively, followed by the sensitivity to be 50. 0% and 88. 9% for the diagnosis of stage Ⅰ-Ⅱand Ⅲ-Ⅳ disease. Conclusion Our resuits showed KLK6 may be one of the reliable indexes for the diagnosis and monitor of ovarian cancer.
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Objective To investigate whether delayed treatment with exogenous kallikrein on neurogenesis after focal cortical infarction in stroke-prone renovascular hypertensive rats (RHRSP). Methods Seventy-two RHRSP were divided into 3 groups. Twenty-four rats were given human tissue kallikrein ( 1.6 × 10-2 PNAU/kg) and 24 rats were given vehicle through tail venous daily for 2 or 6 days consecutively starting at the 24th hour after distal middle cerebral artery occlusion (MCAO). 24 rats underwent sham-operation. Cell proliferation was examined by using 5'-bromo-2'-deoxyuridine (BrdU, 50 mg/kg). Rats were respectively sacrificed 3, 7, 14 or 28 days after MCAO. Results Treatment with kallikrein significantly increased the number of BrdU+ cells in the ipsilateral subventricular zone (SVZ) (304.0±73. 9 vs 167.0±32.2 vs 56.0±12.2 at 7 d after operation, q =7.165, 12.916 and 5.751 respectively,all P<0.05) and in the peri-infarction region (490.0±82.0 vs 308.0±51.5 vs 49.0± 9.5 at 7 d after operation, q = 7.920, 19.184 and 11.264 respectively, all P < 0.01 ), and increased the number of BrdU+/DCX+ cells (225.0±13.6 vs 98.0±9.6 vs 23.0±5.6 at 7 d after operation, q = 30.731,48.735 and 18.004 respectively,all P < 0.01) in the ipsilateral SVZ compared with the vehicle group or the sham-operated group, which began on the 3 day, peaked in 7--14 days after MCAO, and then gradually decreased. Compared with the vehicle group, exogenous kallikrein markedly increased the number of BrdU+/NeuN+ cells (21.0±3.4 vs 13.0±2.6 at 14 d, P =0.001 ) in the peri-infarction region after MCAO. The kallikrein group showed a better functional improvement than the vehicle group after stroke ( all P < 0.05). Conclusion Our study suggests that administration of exogenous kallikrein at 24 h after cortical infarction enhances the SVZ neuroblasts proliferation, migration, and selective differentiation and improves functional recovery after stroke.
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Objective To discuss the mechanism of aprotinin in auxiliary treatment of cerebral hemorrhage.Methods Totally 83 patients were divided into two groups: aprotinin treating group (42 cases) and control group (41 cases). The patients’ neurologic function was marked according to the European Standards of Grading For Cerebral Attacking. The volumes of edema were observed by CT in 10 days through 14 days after their attacks. The patients’ following prothrombin, activated partial thromboplastin time, and fibrinogen values were measured respectively before and after treatment.Results The therapeutic effects in aprotinin treating group(14.5?4.3, 19.0?5.2, 24.0?5.0, respectively) were significantly higher than those in control group(12.7?3.8, 16.8?4.8, 21.7?5.4, respectively).The producing of edema per day (0.212?0.064) in aprotinin treating group was significantly less than that in the control group(0.240?0.063). While the APTT after treatment was significantly longer ((1.2?1.8)s vs (0.4?1.8)s). Conclusions Aprotinin might inhibit the formation of cerebral edema and improve the neurologic function marks after attacks. It should be in treatment better to use aprotinin simultaneously than to use mannitol only. The mechanism properly shown is the preventive effects on bradykinin and thrombin.
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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
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Objective To evaluate the efficacy and safety of a new drug,human urinary kallidinogenase,against acute brain infarction.Method A 15-center,randomized,double-blinded and 3:1 placebo-controlled study was carried out.Acute brain infarction within 48 hours of onset in the territory of the middle cerebral artery were indicated as subjects;kallidinogenase or placebo which was dissolved in 50 ml saline,was slowly injected intraveousely within 30 minutes daily for 3 weeks.The European Stroke Scale and Barthel Index were used to evaluate the neurological deficit and the activities of daily living(ADL),followed by a follow-up at the end of the third month.Results 446 patients were enrolled,who completed ITT analysis,including 330 in kallidinogenase group and 116 in placebo group,meanwhile 421 proceeded with PP analysis(311 and 110 respectively).There were no significant differences of the baseline data between the 2 groups.At the end of treatment,the ESS scores increased by 55.1%?33.0% and 44.7%?32.8% respectively in kallidinogenase group(KG)and placebo group(PG,P=0.0022),the difference being significant.PP analysis had similar results.As for ADL,follow-up 90 days after the treatment showed 374 cases followed,280 in KG and 94 in PG;1 died in PG,while none in KG.In KG,the cases whose BI≥50 were significantly more than those in PG(P=0.0228).Adverse events possibly or definitely attributable to the drug were observed in 27 cases(7.74%),mostly were mild,such as palpitation,flush,dizziness, nausea etc,without special management needed.Only 2 died which was confirmed not correlated to kallidinogenase,and another 2 cases of sudden blood pressure drop were observed.The blood pressure drop, quickly restoring soon after the withdrawal of kallidinogenase and use of hemopiesic drugs,was considered to be caused by the combination use of anti-hypertensive drug ACEI and quick infusion speed.Conclusion Kallidinogenase is efficacious for acute brain infarction in improving the neurological deficits,which is safe in clinical use.