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Objective:To explore and analyze the relationship between serum KLK11 and MK levels and the effect of first iodine 131 ( 131I) ablation after operation for differentiated thyroid cancer. Method:108 patients with differentiated thyroid cancer who underwent total thyroidectomy in our hospital from Jun. 2020 to Jun. 2021 were consecutively selected, and received radioactive ablation after surgery. There were 37 males and 71 females. The age was (48.32±4.25) years, ranging from 28 to 79 years. The patients were divided into successful ablation according to whether the ablation was successful after treatment. There were 64 cases in the group and 44 cases in the unsuccessful group, and 60 healthy people with no abnormality in physical examination during the same period were selected as the control group. The patients were divided into a metastasis-positive group of 20 cases and a metastasis-negative group of 88 cases according to whether lymph nodes occurred. After surgery, serum samples of all subjects were taken, and enzyme-linked immunosorbent assay was used to detect the levels of serum kallikrein-related peptidases 11 (KLK11) and midkine (MK) , and the levels of serum KLK11 and MK were analyzed. Gender, age, BMI, TNM stage, TSH, maximum diameter of lesion, and duration of nail removal were collected. Univariate analysis and logistic regression analysis were used to analyze the independent risk factors of postoperative efficacy.Result:The levels of serum KLK11 and MK in the successful and unsuccessful groups were higher than those in the control group, while the levels of KLK11 and MK in the unsuccessful group were higher than those in the successful group (KLK11: t= 2.642, P<0.05; MK: t=11.906, P<0.05) . The serum levels of KLK11 and MK in the metastasis-positive group were higher than those in the metastasis-negative group (KLK11: t= 2.908, P<0.05; MK: t=14.907, P<0.05) . Univariate analysis showed that BMI ( χ2=6.780, P=0.009) , maximum diameter of lesions ( χ2=14.819, P=0.001) , TSH ( χ2=13.627, P=0.001) , serum KLK11 ( t=2.642, P=0.01) , and serum MK ( t=11.906, P<0.001) were associated with the effect of first 131I ablation after surgery for differentiated thyroid cancer ( P<0.05) . Taking the success of ablation as the dependent variable, a multivariate logistic regression analysis was performed. The results showed that the maximum diameter of the lesions greater than 2 cm ( OR=10.740, 95%CI: 7.033-16.401) , increased level of TSH ( OR=8.559, 95%CI: 2.812-26.057) , increased serum KLK11 level ( OR=16.710, 95%CI: .548-32.666) and increased serum MK level ( OR=10.580, 95%CI: 6.294-17.786) were the factors affecting the first 131I ablation effect after DTC surgery ( P<0.05) . Conclusion:The elevated levels of serum KLK11 and MK are independent risk factors affecting the efficacy of the first 131I ablation after surgery for differentiated thyroid cancer.
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The aim of this study was to investigate the mechanism by which a diet inducing high hyperhomocysteinemia (HHcy) leads to the deterioration of erectile function in rats and whether this is inhibited by expression of the human tissue kallikrein-1 (hKLK1) gene. We established a rat model of HHcy by feeding methionine (Met)-rich diets to male Sprague-Dawley (SD) rats. Male wild-type SD rats (WTRs) and transgenic rats harboring the hKLK1 gene (TGRs) were fed a normal diet until 10 weeks of age. Then, 30 WTRs were randomly divided into three groups as follows: the control (n = 10) group, the low-dose (4% Met, n = 10) group, and the high-dose (7% Met, n = 10) group. Another 10 age-matched TGRs were fed the high-dose diet and designated as the TGR+7% Met group. After 30 days, in all four groups, erectile function was measured and penile tissues were harvested to determine oxidative stress, endothelial cell content, and penis fibrosis. Compared with the 7% Met group, the TGR+7% Met group showed diminished HHcy-induced erectile dysfunction (ED), indicating the improvement caused by hKLK1. Regarding corpus cavernosum endothelial cells, hKLK1 preserved endothelial cell-cell junctions and endothelial cell content, and activated protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) signaling. Fibrosis assessment indicated that hKLK1 preserved normal penis structure by inhibiting apoptosis in the corpus cavernosum smooth muscle cells. Taken together, these findings showed that oxidative stress, impaired corpus cavernosum endothelial cells, and severe penis fibrosis were involved in the induction of ED by HHcy in rats, whereas hKLK1 preserved erectile function by inhibiting these pathophysiological changes.
Subject(s)
Animals , Humans , Male , Rats , Apoptosis , Diet , Endothelial Cells , Erectile Dysfunction/prevention & control , Fibrosis , Hyperhomocysteinemia/complications , Methionine , Oxidative Stress , Penis/pathology , Rats, Sprague-Dawley , Rats, Transgenic , Signal Transduction/genetics , Tissue Kallikreins/geneticsABSTRACT
The aim of this study was to investigate the mechanism by which a diet inducing high hyperhomocysteinemia (HHcy) leads to the deterioration of erectile function in rats and whether this is inhibited by expression of the human tissue kallikrein-1 (hKLK1) gene. We established a rat model of HHcy by feeding methionine (Met)-rich diets to male Sprague-Dawley (SD) rats. Male wild-type SD rats (WTRs) and transgenic rats harboring the hKLK1 gene (TGRs) were fed a normal diet until 10 weeks of age. Then, 30 WTRs were randomly divided into three groups as follows: the control (n = 10) group, the low-dose (4% Met, n = 10) group, and the high-dose (7% Met, n = 10) group. Another 10 age-matched TGRs were fed the high-dose diet and designated as the TGR+7% Met group. After 30 days, in all four groups, erectile function was measured and penile tissues were harvested to determine oxidative stress, endothelial cell content, and penis fibrosis. Compared with the 7% Met group, the TGR+7% Met group showed diminished HHcy-induced erectile dysfunction (ED), indicating the improvement caused by hKLK1. Regarding corpus cavernosum endothelial cells, hKLK1 preserved endothelial cell-cell junctions and endothelial cell content, and activated protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) signaling. Fibrosis assessment indicated that hKLK1 preserved normal penis structure by inhibiting apoptosis in the corpus cavernosum smooth muscle cells. Taken together, these findings showed that oxidative stress, impaired corpus cavernosum endothelial cells, and severe penis fibrosis were involved in the induction of ED by HHcy in rats, whereas hKLK1 preserved erectile function by inhibiting these pathophysiological changes.
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·AIM: To investigate the changes of serum levels of vascular endothelial growth factor ( VEGF ) , Endostatin (ES), thrombospondin (TSP), tissue kallikrein (TKLK) and soluble intercellular adhesion molecule-1 ( sICAM-1) in patients with diabetic retinopathy ( DR ) and its clinical significance.·METHODS:Selected 60 patients with non-proliferative diabetic retinopathy ( NPDR group ) , 60 patients with proliferative diabetic retinopathy ( PDR group ) were enrolled in this study. Sixty diabetic patients without diabetic retinopathy ( DM group ) and 60 healthy people ( control group) were also enrolled. Collection time was from January 2014 to December 2016. Serum levels of VEGF, ES, TSP, TKLK and sICAM-1 were measured and compared.·RESULTS: The levels of serum VEGF, TKLK and sICAM-1 in PDR group were significantly higher than those in NPDR group, DM group and control group ( P<0. 05). The ES of PDR group was significantly lower than that of NPDR group, DM group and control group ( P<0. 05). The levels of VEGF, TKLK and ES in the NPDR group were significantly higher than those in the DM group and the control group (P<0. 05). The serum VEGF in the NPDR group was positively correlated with the levels of ES, TKLK and sICAM-1 (P<0. 05). The serum VEGF of PDR group was positively related to the levels of TKLK and sICAM-1 (P<0. 05). There was no significant relationship between serum VEGF with ES and TSP in PDR group (P>0. 05).·CONCLUSION: The levels of serum ES, TSP, TKLK and sICAM - 1 in patients with DR have changed significantly, and the process of retinopathy has been affected by regulating the level of VEGF.
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OBJECTIVE@#To study the influence of cisplatin implants on transplantation tumor growth and the expression of tissue kallikrein-7 (KLK7) and E-cadherin (E-cad) in tumor-bearing mice with gastric cancer.@*METHODS@#BALB/c nude mice were collected as experimental animal and were randomly divided into model control group (Group A), tail intravenous injection of cisplatin group (Group B), intratumor injection of cisplatin group (Group C) and cisplatin implants treatment group (Group D). After the drugs intervening, the weight and volume of transplantation tumors were measured on Day 20, Day 30 and Day 40 and serum and KLK7 and E-cad contents in transplanted tumor tissue were examined.@*RESULTS@#On Day 20, Day 30 and Day 40 after treatment, the weight and volume of transplantation tumors of tumor-bearing mice in four groups were different (Group A > Group B > Group C > Group D). The contents of KLK-7 and E-cad in tumor tissue and serum of tumor-bearing mice in four groups were different (Group A > Group B > Group C > Group D in KLK-7) and (Group A < Group B < Group C < Group D in E-cad). The weight and volume, and KLK7 and E-cad contents of transplantation tumors in four groups were significant difference (P < 0.05).@*CONCLUSION@#Cisplatin implants can inhibit the growth of transplanted tumor tissue and down-regulated KLK7 expression and up-regulated E-cad expression of tumor-bearing mice with gastric cancer.
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Objective To study the influence of cisplatin implants on transplantation tumor growth and the expression of tissue kallikrein-7 (KLK7) and E-cadherin (E-cad) in tumor-bearing mice with gastric cancer. Methods BALB/c nude mice were collected as experimental animal and were randomly divided into model control group (Group A), tail intravenous injection of cisplatin group (Group B), intratumor injection of cisplatin group (Group C) and cisplatin implants treatment group (Group D). After the drugs intervening, the weight and volume of transplantation tumors were measured on Day 20, Day 30 and Day 40 and serum and KLK7 and E-cad contents in transplanted tumor tissue were examined. Results On Day 20, Day 30 and Day 40 after treatment, the weight and volume of transplantation tumors of tumor-bearing mice in four groups were different (Group A > Group B > Group C > Group D). The contents of KLK-7 and E-cad in tumor tissue and serum of tumor-bearing mice in four groups were different (Group A > Group B > Group C > Group D in KLK-7) and (Group A < Group B < Group C < Group D in E-cad). The weight and volume, and KLK7 and E-cad contents of transplantation tumors in four groups were significant difference (P < 0.05). Conclusion Cisplatin implants can inhibit the growth of transplanted tumor tissue and down-regulated KLK7 expression and up-regulated E-cad expression of tumor-bearing mice with gastric cancer.
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AbstractBackground:Human tissue kallikrein (hK1) is a key enzyme in the kallikrein–kinin system (KKS). hK1-specific amidase activity is reduced in urine samples from hypertensive and heart failure (HF) patients. The pathophysiologic role of hK1 in coronary artery disease (CAD) remains unclear.Objective:To evaluate hK1-specific amidase activity in the urine of CAD patientsMethods:Sixty-five individuals (18–75 years) who underwent cardiac catheterism (CATH) were included. Random midstream urine samples were collected immediately before CATH. Patients were classified in two groups according to the presence of coronary lesions: CAD (43 patients) and non-CAD (22 patients). hK1 amidase activity was estimated using the chromogenic substrate D-Val-Leu-Arg-Nan. Creatinine was determined using Jaffé’s method. Urinary hK1-specific amidase activity was expressed as µM/(min · mg creatinine) to correct for differences in urine flow rates.Results:Urinary hK1-specific amidase activity levels were similar between CAD [0.146 µM/(min ·mg creatinine)] and non-CAD [0.189 µM/(min . mg creatinine)] patients (p = 0.803) and remained similar to values previously reported for hypertensive patients [0.210 µM/(min . mg creatinine)] and HF patients [0.104 µM/(min . mg creatinine)]. CAD severity and hypertension were not observed to significantly affect urinary hK1-specific amidase activity.Conclusion:CAD patients had low levels of urinary hK1-specific amidase activity, suggesting that renal KKS activity may be reduced in patients with this disease.
ResumoFundamento:A calicreína tecidual humana (hK1) é enzima-chave do sistema calicreína-cinina (SCC). A atividade amidásica da hK1 está reduzida na urina de pacientes com hipertensão e insuficiência cardíaca (IC); seu papel na doença arterial (DAC) coronariana ainda não está esclarecido.Objetivo:Avaliar a atividade amidásica da hK1 na urina de pacientes com DAC.Métodos:Sessenta e cinco indivíduos (18 a 75 anos) que se submeteram ao cateterismo cardíaco (CAT) coletaram amostra do jato médio de urina imediatamente antes do CAT. Baseando-se na presença de lesões coronarianas, os pacientes eram classificados em dois grupos: DAC (43 pacientes) e sem DAC (22 indivíduos). A atividade amidásica da hK1 foi estimada com o substrato cromogênico D-Val-Leu-Arg-Nan. Creatinina foi determinada pelo método de Jaffé. A atividade amidásica específica da hK1 urinária foi expressa em µM/(min . mg de creatinina) para corrigir diferenças no fluxo urinário.Resultados:A atividade amidásica da hK1 urinária foi semelhante entre os pacientes com DAC [0,146 µM/(min . mg de creatinina)] e aqueles sem DAC [0,189 µM/(min . mg de creatinina)] (p = 0,803), e permaneceu entre os baixos valores previamente publicados para pacientes com hipertensão primária [0,210 µM/(min . mg de creatinina)] e para aqueles com IC [0,104 µM/(min . mg de creatinina)], respectivamente. Nenhum efeito estatisticamente significativo da gravidade da DAC e da hipertensão sobre a atividade amidásica da hK1 urinária foi observado.Conclusão:A atividade amidásica da hK1 na urina estava reduzida nos pacientes com DAC, o que pode sugerir que a atividade do SCC renal esteja reduzida nessa doença.
Subject(s)
Adolescent , Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , Amidohydrolases/urine , Coronary Artery Disease/urine , Tissue Kallikreins/urine , Biomarkers/urine , Cross-Sectional Studies , Coronary Artery Disease/physiopathology , Creatinine/urine , Heart Failure/physiopathology , Heart Failure/urine , Hypertension/physiopathology , Hypertension/urine , Kallikrein-Kinin System/physiology , Reference Values , Severity of Illness Index , Statistics, NonparametricABSTRACT
Objective It remains to be confirmed whether tissue kallikrein has neuroprotective effect in diabetes-induced stroke.This study was to investigate the neuroprotection of tissue kallikrein against cerebral ischemia-reperfusion injury in diabetic rats. Methods Healthy male SD rats were randomly divided into a sham operation, a saline control, and a tissue kallikrein group.Diabetes mellitus was induced in the animals by intraperitoneal injection of streptozotocin and the model of focal cerebral ischemia-reperfusion was made with an intraluminal vascular occlusion method. At 24 hours after modeling, we obtained the neurological deficit score, in-farct size, and brain water content, counted Iba1-and MPO-positive cells by immunohistochemistry, and determined the expressions of ICAM-1 and VCAM-1 by real-time PCR. Results In comparison with the saline controls, the rats treated with tissue kallirein showed significant decreases in the neurological deficit score (P<0.01), the infarct size ([23.57 ±5.79] vs [47.97 ±1.19]%, P<0.01), brain edema ([81.73 ±2.10] vs [84.94 ±2.34]%, P<0.05), the counts of Iba1-and MPO-positive cells (12.33 ±4.46 vs 31.83 ±8.13 and 13.83 ±4.49 vs 37.50 ±7.64, both P<0.01), and the expressions of ICAM-1 and VCAM-1 (both P<0.05). Conclusion Tissue kallikrein has a neuroprotective effect against cerebral ischemia-reperfusion injury in diabetic rats, which may be associated with its anti-inflammation property.
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Kallikrein-kinin system consists of kininogen , kallikrein, bradykinin and kinin .Kinins, derived from kininogen by tissue kallikrein , play their biological effects via bradykinin 1/2 receptors or protease activated receptors .Existing researches suggest that kinins exert various effects through different intracellular and mitochondrial signal pathways such as MAPK , PI3K/Akt/GSK3 be-ta, NO, JAKs/STATs.This review aims to elucidate the roles and the intracellular signal pathways of KKS in different diseases .
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The human tissue kallikrein(KLK)gene family consists of 15 highly conservative serine pro-teases,which is the largest uninterrupted cluster of protease genes in the human genome. Several members of the family are expected to be markers for tumor diagnosis and prognosis. Studies find that the expressions of KLK2-11 and KLK13-15 are abnormal,and the majority of KLKs have potential diagnostic and prognostic values.
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BACKGROND: Low level laser therapy (LLLT) has been used clinically in order to treat inflammation, where tissue and plasma prekallikrein have crucial importance. Plasma prekallikrein (PPK) is synthesized by the hepatocytes and secreted into the bloodstream, where it participates in the surface-dependent activation of blood coagulation, fibrinolysis, kinin generation and inflammation. Tissue prekallikrein is associated with important disease states (including cancer, inflammation, and neurodegeneration) and has been utilized or proposed as clinically important biomarker or therapeutic target of interest. OBJECTIVE: To evaluate if LLLT modulates tissue and plasma prekallikreins mRNA expression in the carrageenan-induced rat paw edema. METHODS: Experimental groups were assigned as followed: A1 (Control-saline), A2 (Carrageenan-only), A3 (laser 660nm only) and A4 (Carrageenan + laser 660nm). Edema was measured by a plethysmometer. Subplantar tissue was collected for the quantification of prekallikreins mRNA by Real time-Polymerase Chain Reaction. RESULTS: A significantly decrease in the edema was observed after laser irradiation. Expression of prekallikreins increased after carrageenan injection. Tissue and plasma prekallikrein mRNA expression significantly decreased after LLLT's 660nm wavelength. CONCLUSION: These results suggest that expression of tissue and plasma prekallikreins is modulated by LLLT, which can be used in clinical practice due to its anti-inflammatory effects.
CONTEXTUALIZAÇÃO: A laserterapia de baixa potência tem sido usada para o tratamento de processos inflamatórios diversos em que a calicreína tecidual e a plasmática possuem participação ativa. A pré-calicreína plasmática (PPK) é sintetizada pelos hepatócitos e secretada na corrente sanguínea, onde participa da ativação da coagulação, fibrinólise, geração de cininas e inflamação. A pré-calicreína tecidual está associada com importantes doenças (incluindo câncer, inflamação e neurodegeneração) e tem sido utilizada ou sugerida clinicamente como importante biomarcador ou alvo terapêutico. OBJETIVO: Avaliar se a laserterapia altera a expressão gênica da pré-calicreína tecidual e da plasmática no modelo de inflamação aguda induzida pela carragenina. MÉTODOS: Quarenta ratos foram separados em quatro grupos experimentais: A1 (controle), A2 (carragenina, apenas), A3 (laser 660nm, apenas) e A4 (Carragenina + laser 660nm). O edema foi medido por um pletismômetro. Tecido subplantar foi coletado para a quantificação de RNA mensageiro (RNAm) de pré-calicreínas tecidual e plasmática por PCR em tempo real. RESULTADOS: Observou-se uma diminuição significativa no volume de edema após irradiação com laser 660nm. A expressão de RNAm de pré-calicreínas tecidual e plasmática aumentou após a inoculação de carragenina, entretanto a expressão gênica das pré-calicreínas diminuiu significantemente após laserterapia de baixa potência de 660nm. CONCLUSÃO: Os resultados sugerem que a expressão de RNAm das pré-calicreínas tecidual e plasmática é modulada pela laserterapia de baixa potência, podendo ser alvo terapêutico para tratamento de processos inflamatórios.
Subject(s)
Animals , Male , Rats , Edema/radiotherapy , Low-Level Light Therapy , Prekallikrein/biosynthesis , Prekallikrein/genetics , RNA, Messenger/biosynthesis , Carrageenan/administration & dosage , Disease Models, Animal , Extremities , Edema/blood , Edema/chemically induced , Rats, Wistar , RNA, Messenger/bloodABSTRACT
Objective To prepare monoclonal antibody(McAb) against human tissue kallikrein (HK) and develop an ELISA kit allows for the in vitro quantitative determination of human tissue kallikrein in urine. Methods To generate a monoclonal antibody specific for TK, the synthetic TK peptide consisting of 12 amine acids(12P), was fused to keyhole limpet hemocyanin(KLH) and used for immunization. Using hybridoma screening, monoclonal secreting cell lines were identified and used to generate ascites in BALB/c mouse. Antibody was purified by affinity column chromatography. 12% SDS-PAGE and Western blot were used to visualize the purified antibody. This kit employs indirect competitive ELISA technique and BiotinAvidin System. 12P was fused to bovine serum albumin(BSA) and has been pre-coated onto a microplate at first. Standards and samples were added to the appropriate microplate wells with a biotin-conjugated McAb croplate well. A TMB substrate solution is added to each well. The enzyme-substrate reaction is terminating by the addition of a sulphuric acid solution and the color change is measured spectrotometrically at a wavelength of 450 nm. The concentration of tissue kallikrein in the samples is then determined by comparing the O.D. of the samples to the standard curve. Results 8 hybridoma cell lines secreting mAbs special to HK,SDS-PAGE and Western blot demonstrated successful preparing and purification of McAb( 100% ). The linearity of this ELISA kit is demonstrated(r =0. 990). The range of detection of the assay is 0.008 μg/ml to 0. 5 μg/ml. The assay remained stable, with no change in the values measured, over five cycles of freezing and thawing. Conclusion 8 McAbs against HK have been prepared successfully and possess high titer and specificity. The development of an ELISA kit for detecting HK can meet the needs of detection of HK in urine samples.
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ObjectiveTo investigate the diagnostic and prognostic significance of serum human kallikrein11 ( KLK11 ) in patients with ovarian cancer. MethodsThe level of serum KLK11 was determined by enzyme-linked immunoassay(ELISA) for 30 patients with ovarian cancer, 26 patients with benign gynecologic processes and 32 healthy subjects,and the KLK11 value beyond the upper-limit of 95％ credibility interval( 95％ CI) based on the health controls was used as the positive value. The positive rate of KLK11 was compared among the three groups.ResultsThe positive rate of KLK11 in patients with ovarian cancer was significantly higher than those in patients with benign gynecologic processes or in healthy subjects[(73.3％) and(7.7％ ) or(6.3％ ) ,all P ＜0.01 )] ,and there was no significant difference between ovarian benign tumor group and the healthy control group ( P ＞ 0.05 ).ConclusionThe serum KLK11 should be a novel tumor marker in the clinical diagnosis of ovarian cancer.
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Response surface methodology was undertaken to optimize the polyethylenimine-mediated transient transfection of suspension cultured HEK 293-F cells. A total of 15 combinations were designed according to Box-Behnken design to identify the effects of DNA concentration, polyethylenimine concentration and incubation time on transient transfection efficiency. The highest integral optic density of green fluorescent protein presenting r-protein yield was accessed using a DNA concentration of 1.75 ug/mL, a polyethylenimine concentration of 10.5 ug/mL, and an incubation time of 11.8 min. Analysis of variance demonstrated that the experimental values fit well with a quadratic model. The RSM-optimized transient transfection resulted in greater production of human tissue prokallikrein (TproK) than non-RSM-optimized conditions: protein yield was 32.0 mg/L and the maximum viable cell density reached 3.57 x 10(6) cells/mL in a 5 L stirred-tank bioreactor culture.
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Humans , Tissue Kallikreins/genetics , Gene Expression , Transfection , Analysis of Variance , Bioreactors , Cell Line , Polyethyleneimine , Genetic Vectors/geneticsABSTRACT
AIM: To investigate the effects of adenovirus-mediated human tissue kallikerin (Ad-hKLK1) gene delivery on the proliferation, migration of VSMC_(SHR) induced by platelet derived growth factor-BB (PDGF-BB). METHODS: The VSMC_(SHR) proliferation induced by PDGF-BB was accessed by cell counting and methyl thiazolyl tetrazoliuin (MTT). The migration was assessed by modified Boyden chamber assay. Western blotting was used to determine the expressions of the cycle-independent kinase inhibitors p27~(Kip1) and p21~(Cip1).RESULTS: Proliferation of VSMC_(SHR) induced by PDGF-BB was inhibited after transfection of Ad-hKLK1 (20-100 MOI) in a MOI-dependent manner. The peak inhibition titer of Ad-hKLK1 fell on 100 MOI, with the peak inhibition rate of 39.3% (cell counting, n=3, P<0.01), 30.2% (MTT, n=3, P<0.01), peak stunning rate of cell-cycle in phase G0/G1 at 36.4%. The inhibitory effects of proliferation and cell-cycle caused by hKLK1 gene delivery were significantly abolished by Hoe140, a bradykinin B2 receptor antagonist. Migration of VSMC_(SHR) induced by PDGF-BB was inhibited after hKLK1 gene delivery, with the peak inhibitory rate of 34.6% (n=6, P<0.01). However the inhibitory effects of migration were not blocked by Hoe140. The protein expression of p27~(Kip1) and p21~(Cip1) increased significantly after the hKLK1 gene delivery, whereas Hoe140 nearly completely blocked these effects (n=3, P<0.01, respectively).CONCLUSION: The hKLK1 gene delivery may inhibit the proliferation and migration of VSMC_(SHR) induced by PDGF-BB. Bradykinin B2 receptor probably mediates the up-regulating expression of p27~(Kip1) and p21~(Cip1) that contributes to the inhibitory effects of proliferation of hKLK1. However, the inhibitory effects of migration by hKLK1 gene delivery may not be mediated by bradykinin B2 receptor.
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Objective To evaluate the therapeutic effect of recombinant adeno-associated viral vector (rAAV) expressing human tissue kallikrein gene (rAAV-HK) on insulin resistance and renal complications in tyDe2 diabetic rats. Methods Male Wistar rats were injected low dose streptozotocin and fed with high fat and sucrose diets to form type 2 diabetic model. rAAV mediated HK gene (HK group) or LacZ gene (LacZ group) were introduced to the diabetic rats, and their systolic blood pressure, fasting blood glucose and insulin, serum creatinine, urine creatinine, urine osmolarity and urine microalbumin were measured. The homeostasis model assessment of insulin resistance (HOMA-IR), urinary albumin excretion rate (UAER) and creatinine clearance rate (Ccr) were calculated. The expression of PI3-kinase p11o catalytic subunit (p110) and Akt phosphorylation on Thr-308 were detected by Western blot. The morphology of kidney wag observed. Results Delivery of rAAVHK resulted in a reduction in blood pressure at 2 weeks and the hypotensive effect lasted for the duration of the study. The HOMA-IR was significantly lower in HK group than LacZ group (4.76±0.33 vs 8.36±0.48, P<0.01) at the end of the study, fasting insulin level was reduced [(8.19±2.45 vs 13.85±3.76)mIU/L. P<0.01], but there was no significant change in fasting blood glucose [(13.09±3.01 vs 13.58±2.88)mmol/L].The phosphorylation of p11o and Akt Thr-308 were significantly decreased in skeletal muscle and liver in LacZ group and were almost corrected by HK gene therapy. The UAER and Ccr were significantly lower and urinary osmolarity were higher in HK-treated rats compared with LaeZ rats. Histological assessment indicated that the renal complication was relieved by HK gene delivery. Conclusion The rAAV-mediated HK gene delivery efficiently attenuated insulin resistance partly through PI3K/Akt pathway and diabetic nephropathy in type 2diabetic rats.
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To evaluate the feasibility of treating hypertension by human tissue kallikrein gene (KLK1) delivery and by enzyme (rK1) administration, two recombinant vectors expressing KLK1 cDNA were constructed for gene delivery (pcDNA-KLK1) and recombinant enzyme preparation (pOV-KLK1). Expression of the pcDNA-KLK1 vector in COS-1 cells was confirmed by immunofluorescence and in spontaneous hypertension rats (SHR) by enzymatic detection. Following intramuscular or intravenous injection with the pcDNA-KLK1 vector, systolic pressure of SHR was significantly decreased, which lasted for 20 d to two months depending on dose, route and/or time of injection. Egg white containing recombinant hK1 was prepared by injection of egg-laying hens with the oviduct-specific expression vector pOV-KLK1 and administered into SHR via oral gavage. Following administration, systolic pressure of the SHR was decreased to that of normal rats, which lasted for 3-5 d depending on the dosage used. These data suggest that both hKLK1 gene delivery and recombinant enzyme administration can be used as alternative strategies for treating human hypertension.
Subject(s)
Animals , Female , Humans , Rats , Blood Pressure/physiology , COS Cells , Chlorocebus aethiops , Chickens , Genetic Therapy , Gene Transfer Techniques , Genetic Vectors/genetics , Hypertension/genetics , Hypotension/genetics , Rats, Inbred SHR , Recombinant Proteins/administration & dosage , Tissue Kallikreins/geneticsABSTRACT
Objective To explore the therapeutical effect and its mechanism of tissue kallikrein (TK) on cerebral ischemic-reperfusion rat.Methods 36 rats were randomly divided into three groups: sham operation group, NS group [administrated with normal sodium 2 ml/(kg?d)], TK group [administrated with TK 17.5?10-3U/(kg?d)]. The NS and TK groups rats were injected intraperitoneally with NS or TK for three consecutive days. After that, neurological function and the infarct volume was appraised in each of group. Meanwhile, the ischemic tissue were detected by Nissl's staining. TUNEL staining and immunohistochemistry were used to examine the caspase-3,TNF-?,IL-1,NSE,GFAP,vWF expression in ischemic tissue.Results Compared with NS group, TK group significantly reduced the loss of neurological function and the infarct volume and improved the change of pathology in ischemic tissue(all P