Strength training on soccer players' kicking stability / Treinamento de força na estabilidade nos chutes dos jogadores de futebol / Entrenamiento de fuerza en la estabilidad de las patadas de los futbolistas

ABSTRACT Introduction: The physical condition of soccer players in sports competitions has improved over the years. The optimal performance of their professional skills in competitive conditions has become essential for victory in soccer matches. Objective: This paper explores the effects of different methods employing strength training on soccer kicking techniques aiming at the set that best enables the accuracy of hits through its stability. Methods: 36 soccer players were randomly divided into the experimental and control groups, with no statistical difference in fitness and the comprehensive ability characteristics of the players. Both subjects were trained for 12 weeks; only the experimental group received the special strength training intervention for stability. The passing score of the curve ball in 20-meter dribbling was measured before and after training. The data were statistically treated. Results: The kicking accuracy of soccer players in the experimental group differed from before the test (P<0.01). There was also a significant difference in kicking accuracy in the control group (P<0.05). The 20-meter arc dribbling scores in the experimental group were statistically significant compared to those before the test (P<0.05). There was no significant difference between the control group and the test scores on curve ball passing scores in 20-meter dribbling (P>0.05). Conclusion: Functional strength methods to achieve the goal of improving kicking accuracy in athletes have been developed. Coaches should pay attention to physical training, an attitude that encourages players to achieve sufficient physical strength for soccer games with their kicking skills. Level of evidence II; Therapeutic studies - investigation of treatment outcomes.

RESUMO Introdução: A condição física dos jogadores de futebol nas competições esportivas tem se aprimorado ao longo dos anos e o desempenho ótimo de suas habilidades profissionais em condições competitivas tornou-se um fator essencial para a vitória nas partidas de futebol. Objetivo: Este artigo explora os efeitos de diferentes métodos empregando o treinamento de força sobre as técnicas de chute de futebol visando o conjunto que melhor capacita a precisão de acertos através de sua estabilidade. Métodos: 36 jogadores de futebol foram divididos aleatoriamente, sem diferença estatística de aptidão física e as características de capacidade abrangente dos jogadores, em grupo experimental e controle. Ambos os grupos de sujeitos foram treinados durante 12 semanas, somente o grupo experimental recebeu a intervenção especial de treinamento de força para estabilidade. A pontuação de passe da bola curva em drible de 20 metros foi medida antes e depois do treino. Os dados foram tratados estatisticamente. Resultados: A precisão do chute dos jogadores de futebol no grupo experimental foi diferente daquela antes do teste (P<0,01). Também houve uma diferença significativa na precisão de chutes no grupo de controle (P<0,05). As pontuações dos dribles de arco de 20 metros no grupo experimental foram estatisticamente significativas em comparação com aquelas antes do teste (P<0,05). Não houve diferença significativa entre o grupo de controle e os resultados do teste na pontuação de passe da bola curva em drible de 20 metros (P>0,05). Conclusão: Métodos de força funcional para alcançar o objetivo de melhorar a precisão de chute nos atletas foram desenvolvidos. Os treinadores devem prestar atenção ao treinamento físico, atitude que incentiva aos jogadores atingirem a força física suficiente para os jogos de futebol com suas habilidades de chute. Nível de evidência II; Estudos terapêuticos - investigação dos resultados do tratamento.

RESUMEN Introducción: La condición física de los futbolistas en las competiciones deportivas ha ido mejorando a lo largo de los años y el rendimiento óptimo de sus habilidades profesionales en condiciones competitivas se ha convertido en un factor esencial para la victoria en los partidos de fútbol. Objetivo: Este trabajo explora los efectos de diferentes métodos que emplean el entrenamiento de la fuerza en las técnicas de pateo de fútbol con el objetivo de encontrar el conjunto que mejor permita la precisión de los golpes a través de su estabilidad. Métodos: Se dividieron aleatoriamente 36 jugadores de fútbol, sin diferencias estadísticas en cuanto a la aptitud física y las características de capacidad integral de los jugadores, en grupo experimental y grupo de control. Ambos grupos de sujetos fueron entrenados durante 12 semanas, sólo el grupo experimental recibió la intervención especial de entrenamiento de fuerza para la estabilidad. Se midió la puntuación del pase de la pelota curva en el regateo de 20 metros antes y después del entrenamiento. Los datos fueron tratados estadísticamente. Resultados: La precisión de las patadas de los futbolistas del grupo experimental fue diferente a la de antes de la prueba (P<0,01). También hubo una diferencia significativa en la precisión de las patadas en el grupo de control (P<0,05). Las puntuaciones del regateo en arco de 20 metros en el grupo experimental fueron estadísticamente significativas en comparación con las anteriores a la prueba (P<0,05). No hubo diferencias significativas entre el grupo de control y las puntuaciones de la prueba en las puntuaciones de los pases de balón curvo en el regateo de 20 metros (P>0,05). Conclusión: Se han desarrollado métodos de fuerza funcional para lograr el objetivo de mejorar la precisión de las patadas en los atletas. Los entrenadores deben prestar atención a la preparación física, una actitud que anima a los jugadores a conseguir la fuerza física suficiente para los partidos de fútbol con sus habilidades de pateo. Nivel de evidencia II; Estudios terapéuticos - investigación de los resultados del tratamiento.

Analysis of drug resistance and risk factors of multi drug resistant bacteria in bloodstream infection / 中国热带医学

@#Abstract: Objective To analyze the antimicrobial resistance rate and risk factors of multi drug resistant organisms (MDRO) in bloodstream infection for rational treatment. Methods　A total of 696 cases of bloodstream infections of Staphylococcus aureus, Enterococcus, Enterobacteriaceae (excluding Salmonella and Shigella), Pseudomonas aeruginosa and Acinetobacter in our hospital from 2017 to 2021 were selected, and 711 pathogenic strains were isolated from their whole blood samples. The antimicrobial resistance rates of various multi drug resistant strains were analyzed and the risk factors of MDRO infection were analyzed. Results　696 non repeated cases were screened out from 13 187 whole blood culture specimens, with a positive rate of 5.3%, and a total of 711 blood influenza pathogens were detected, among them, 350 strains of MDRO were detected with a detection rate of 49.23% (350/711). Among the pathogenic bacteria of bloodstream infection, Escherichia coli was the most, with 277 strains accounting for 38.96% (277/711), of which 201 strains were MDRO, accounting for 57.43% (201/350); followed by Klebsiella pneumoniae and Staphylococcus aureus, with 155 strains accounting for 21.80% (155/711) and 89 strains accounting for 12.52% (89/711), among which 43 strains of Klebsiella pneumoniae MDRO accounted for 12.29% (43/350) and 38 strains of Staphylococcus aureus MDRO accounted for 10.86% (38/350). The change trend of the three pathogens during 2017-2021 was not obvious. The drug sensitivity test showed that Escherichia coli and Klebsiella pneumoniae were highly resistant to cephalosporins and fluoroquinolones, and the drug resistance rate of aminoglycosides was relatively low. They had almost no resistance to cephalosporins and carbapenems. Staphylococcus aureus has a high resistance rate to lincomycin and macrolides, but no resistance to oxazolidinone, glycopeptides and glycylcyclins. There were 350 cases of MDRO infection and 361 cases of non MDRO infection. Univariate analysis showed that the age, sex, cardiovascular and cerebrovascular history, renal insufficiency, lung disease, hypoalbuminemia, hepatobiliary disease, electrolyte disorder and anemia of the patients had no statistical significance in MDRO infection (P>0.05); diabetes, urinary tract infection, surgical operation and burn were the influencing factors of MDRO (P<0.05). According to logistic analysis, diabetes, urinary tract infection, surgical operation and burn were the risk factors of MDRO infection (P<0.05). Conclusion　The infection of MDRO in patients with bloodstream infection is serious, and early prevention and control should be paid attention to, and the principle of graded use of antibiotics should be strictly observed, and the rational application should be carried out to actively and effectively control the production of MDRO.

The effect of PD-1/PD-L1 signaling axis on the interaction between CD19+B cells and CD4+T cells in peripheral blood of patients with systemic lupus erythematosus

Abstract Background The defect of B cell self-tolerance and the continuous antigen presentation by T cells (TCs) mediated by autoreactive B cells (BCs) play a key role in the occurrence and development of systemic lupus erythematosus (SLE). PD-1/PD-L1 signaling axis negatively regulates the immune response of TCs after activation and maintains immune tolerance. However, the effect of PD-1/PD-L1 signaling axis on the interaction between CD19+B/CD4+TCs in the peripheral blood of patients with SLE has not been studied in detail. Methods PD-1/PD-L1 and Ki-67 levels in peripheral blood (PB) of 50 SLE patients and 41 healthy controls (HCs) were detected through flow cytometry, and then the expression of PD-1+/−cells and PD-L1+/−cells Ki-67 was further analyzed. CD19+B/CD4+TCs were separated for cell culture and the supernatant was collected to determine proliferation and differentiation of TCs. IL-10 and IFN-γ secretion in the supernatant was also determined using ELISA. Results The PD-1, PD-L1, and Ki-67 levels on CD19+B/CD4+TCs in patients with SLE were higher than HCs. In CD19+B/CD4+TCs of SLE patients, the proliferative activity of PD-L1+ cells was higher than that of PD-L1− cells, and the proliferative activity of PD-1+ cells was higher than that of PD-1− cells. In the system co-culturing CD19+B/CD4+TCs from HCs/SLE patients, activated BCs promoted TCs proliferation and PD-L1 expression among TCs. Addition of anti-PD-L1 to co-culture system restored the proliferation of TCs, and inhibited IL-10/IFN-γ level. The addition of anti-PD-L1 to co-culture system also restored Tfh and downregulated Treg in HCs. Conclusions Axis of PD-1/PD-L1 on CD19+B/CD4+TCs in PB of SLE patients is abnormal, and cell proliferation is abnormal. In CD19+B/CD4+TCs of SLE patients, the proliferative activity of PD-L1+ and PD-1+ cells compared with PD-L1− and PD-1− cells in SLE patients, respectively. CD19+B/CD4+TCs in SLE patients can interact through PD-1/PD-L1.

Two methods of treatment for skeletal Class Ⅲ malocclusion on airway changes before and after clinical research / 口腔疾病防治

Objective @# To investigate the changes in the sagittal diameter of the upper airway before and after the treatment of skeletal Class Ⅲ malocclusion in adults with microimplant anchorage and class Ⅲ intermaxillary elastics and to provide a reference for clinical treatment. @*Methods @#A total of 35 adult patients with skeletal Class Ⅲ malocclusion were selected to be treated with the straight-wire technique. Microimplant group, 15 cases (group A): patients with severe skeletal Class Ⅲ malocclusion (vertical high angle) were treated with the straight-wire technique combined with microimplant anchorage; class Ⅲ intermaxillary elastics group, 20 cases (group B): Patients with mild or moderate skeletal Class Ⅲ malocclusion (vertical low angle and average angle) were treated with the straight-wire technique combined with class Ⅲ intermaxillary elastics, and cephalometric radiographs obtained before and after treatment in the upper airway in the two groups were measured and analyzed.@*Results @#Changes in cranial and maxillofacial measurements after correction: in group A, (sella-nasion-supramental angle) the SNB angle decreased significantly (P < 0.05), and (subspinale-nasion-supramental angle) the ANB angle increased significantly (P < 0.05). In group B, the SNB angle decreased significantly (P < 0.05), while (subspinale-nasion-subspinale angle) the SNA angle、ANB angle and anterior skull base plane-mandibular plane angle (Sn-MP) angle increased significantly (P < 0.05). Changes in sagittal diameter of the upper airway measurements after corrections: In group A, the width of the glossopharyngeal segment of the upper airway (TB-TPPW) decreased significantly (P < 0.05). In group B, first segment width of the upper airway behind the hard palate (PNS-R) increased significantly (P < 0.05). After correction, the decreased SNB and increased ANB in group A was higher than that in group B, and the difference was statistically significant （P < 0.05）. The decreased of TB-TPPW in upper airway of group A was greater than that of group B, and the difference was statistically significant （P < 0.05）.@* Conclusions @#In the treatment of skeletal class Ⅲ malocclusion with microimplant anchorage, the sagittal diameter of the glossopharyngeal segment of the upper airway has a negative impact.

Expression of TAGLN in colorectal cancer tissues and its effect on malignant biological behaviors of SW480 cells / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the expression of transgelin (TAGLN) in colorectal cancer (CRC) tissues and its effect on the proliferation, migration and invasion of CRC SW480 cells. Methods: Surgically resected CRC tissues and corresponding para-cancerous tissues of 97 CRC patients from May 2015 to August 2016 in the Affiliated Tumor Hospital of Zhengzhou University were collected; In addition, CRC cell lines SW620, SW480, HCT116 and normal colorectal mucosal cell line FHC were also collected for this study. Immunohistochemical staining was used to detect the expression of TAGLN in CRC tissues, and the correlation between TAGLN and patients’ clinicopathological features was analyzed. Quantitative Real-time quantitative polymerase chain reaction (qPCR) and Western blotting (WB) were used to detect the mRNA and protein expressions of TAGLN in CRC cell lines. si-TAGLN and si-Ctrl were respectively transfected into SW480 cells by liposome transfection method. The effects of silencing TAGLN on the proliferation, migration and invasion of SW480 cells were detected by CCK-8, Wound-healing assay and Transwell assay, respectively; and the expression of EMT-related proteins E-cadherin, N-cadherin and vimentin were detected by WB. Results: The positive expression rate of TAGLN in CRC tissues was significantly higher than that in para-cancerous tissues (P<0.01), and TAGLN expression was correlated with TNM stage, degree of tumor differentiation and lymph node metastasis in CRC patients (P<0.05 or P<0.01). The mRNA and protein expression levels of TAGLN in SW480 cells were significantly higher than those in FHC cells (all P<0.01). After TAGLN silence, the proliferation, invasion and migration ability of SW480 cells were significantly reduced (all P<0.01), the expression level of E-cadherin in SW480 cells was increased, while the expression levels of N-cadherin and vimentin were decreased (all P<0.01). Conclusion: TAGLN is highly expressed in CRC tissues and cells. Silencing TAGLN can inhibit the proliferation, invasion and migration of CRC cells, suggesting that TAGLN plays an important role in the occurrence and development of CRC.

Azeliragon ameliorates Alzheimer's disease via the Janus tyrosine kinase and signal transducer and activator of transcription signaling pathway

OBJECTIVES: TTP488, an antagonist of the receptor for advanced glycation end-products, was evaluated as a potential treatment for patients with mild-to-moderate Alzheimer's disease (AD). However, the mechanism underlying the protective action of TTP488 against AD has not yet been fully explored. METHODS: Healthy male rats were exposed to aberrant amyloid β (Aβ) 1-42. Lipopolysaccharide (LPS) and the NOD-like receptor family pyrin domain containing 1 (NLRP1) overexpression lentivirus were injected to activate the NLRP1 inflammasome and exacerbate AD. TTP488 was administered to reverse AD injury. Finally, tofacitinib and fludarabine were used to inhibit the activity of Janus tyrosine kinase (JAK) and signal transducer and activator of transcription (STAT) to prove the relationship between the JAK/STAT signaling pathway and TTP488. RESULTS: LPS and NLRP1 overexpression significantly increased the NLRP1 levels, reduced neurological function, and aggravated neuronal damage, as demonstrated by the impact latency time of, time spent by, and length of the platform covered by, the mice in the Morris water maze assay, Nissl staining, and immunofluorescence staining in rats with AD. CONCLUSIONS: TTP488 administration successfully reduced AD injury and reversed the aforementioned processes. Additionally, tofacitinib and fludarabine administration could further reverse AD injury after the TTP488 intervention. These results suggest a new potential mechanism underlying the TTP488-mediated alleviation of AD injury.

Clinical efficacy of Mohs surgery combined with topical photodynamic therapy for facial basal cell carcinoma

Purpose: The purpose of this study is to investigate the clinical efficacy of Mohs surgery in combination with topical photodynamic therapy (PDT) for facial basal cell carcinoma. Patients and Methods: Eighty-six patients with facial basal cell carcinoma treated in our department from April 2011 to December 2013 were included. Mohs surgery was used to remove the lesions followed by direct suturing, skin flap grafting, or medium thickness free-skin grafting to repair the incisions. Topical PDT was performed three times, at an interval of 2 weeks, immediately after the sutures were removed. The patients were followed up for 2 years after the operation to evaluate tumor recurrence. Results: Recurrence was not observed within 1 year after Mohs surgery combining PDT; however, one case of recurrence was found at the 2-year follow-up. Conclusion: The efficacy of Mohs surgery combining topical PDT is a definite treatment for facial basal cell carcinomas, as it reduced the tumor recurrence rate and maintained the relative integrity of the local tissues and appearance. This method could be a new effective treatment method for the facial basal cell carcinoma

Id-1 expression in colorectal adenocarcinoma tissues and its clinical significance

SUMMARY BACKGROUND: This study aims to investigate the expression of Id-1 in human colorectal adenocarcinoma tissues and explore its correlation with the clinical pathological parameters of colorectal cancer. METHODS: The Id-1 mRNA and protein expression levels of 50 specimens of normal colorectal tissues and 50 specimens of colorectal adenocarcinoma tissues were detected using reverse-transcription polymerase chain reaction and western blot. Furthermore, Id-1 protein was detected using immunohistochemistry. The correlation between the expression of Id-1 and clinicopathologic features was analyzed. RESULTS: The mRNA expression level of Id-1 in colorectal adenocarcinoma tissues and normal colorectal tissues was 0.96 ± 0.03 vs. 0.20 ± 0.04, respectively; and the difference was statistically significant (P=0.011). Furthermore, Id-1 protein expression was higher in colorectal adenocarcinoma tissues than in normal colorectal tissues (0.82 ± 0.04 vs. 0.31 ± 0.02, P=0.020). In addition, the positive protein expression rate of Id-1 was higher in colorectal adenocarcinoma tissues than in normal colorectal tissues (72.00% vs. 24.00%, X2=23.431, P=0.000). The expression of Id-1 was correlated with the depth of tumor invasion, TNM stage, lymph node metastasis, vessel invasion, and liver metastasis (P<0.01). However, this expression was not correlated with tumor size and differentiation degrees (P>0.05). CONCLUSIONS: The high Id-1 expression in colorectal adenocarcinoma tissues play an important role in the process of cancer, and is expected to become a new tumor monitoring indicator for clinical diagnosis, treatment, and prognosis judgment.

RESUMO OBJETIVO: O objetivo deste estudo é investigar a expressão de Id-1 em tecidos de adenocarcinoma colorretal em humanos e investigar sua correlação com os parâmetros patológicos clínicos de câncer colorretal. MÉTODOS: Os níveis de expressão de proteína e mRNA Id-1 em 50 amostras de tecido colorretal normal e 50 amostras de tecido de adenocarcinoma colorretal foram detectados através de reação em cadeia de polimerase precedida de transcrição reversa e western blot. Além disso, a proteína Id-1 foi detectada através de imuno-histoquímica. A correlação entre a expressão de Id-1 e características clínico-patológicas foi analisada. RESULTADOS: O nível de expressão de mRNA Id-1 em tecidos de adenocarcinoma colorretal e tecidos colorretais normais foi de 0,96 ± 0,03 versus 0,20 ± 0,04, respectivamente; a diferença foi estatisticamente significativa (P= 0,011). Além disso, a expressão da proteína Id-1 foi maior em tecidos de adenocarcinoma colorretal do que em tecidos colorretais normais (0,82 ± 0,04 versus 0,31 ± 0,02, P= 0,020). Além disso, a taxa de expressão positiva de proteínas Id-1 foi maior em tecidos de adenocarcinoma colorretal do que em tecidos colorretais normais (72,00% vs. 24,00%, X2=23,431, p=0,000). A expressão de Id-1 foi correlacionada com a profundidade da invasão tumoral, estágio TNM, metástases linfonodais, invasão vascular e metástase hepática (P<0,01). Todavia, essa expressão não se correlacionou com o tamanho do tumor e graus de diferenciação (P>0,05). CONCLUSÃO: A alta expressão de Id-1 em tecidos de adenocarcinoma colorretal desempenham um importante papel no processo do câncer, e é esperado que se torne um novo indicador de monitoramento de tumores para o diagnóstico clínico, tratamento e estimativa de prognóstico.

Analysis of mRNA expression profiles of carotenogenesis and astaxanthin production of Haematococcus pluvialis under exogenous 2, 4-epibrassinolide (EBR)

The fresh-water green unicellular alga Haematococcus pluvialis is known to accumulate astaxanthin under stress conditions. In the present study, transcriptional expression of eight genes involved in astaxanthin biosynthesis exposed to EBR (25 and 50 mg/L) was analyzed using qRT-PCR. The results demonstrated that both 25 and 50 mg/L EBR could increase astaxanthin productivity and the eight carotenogenic genes were up-regulated by EBR with different expression profiles. Moreover, EBR25 induction had a greater influence on the transcriptional expression of ipi-1, ipi-2, crtR-B, lyc and crtO (> 5- fold up-regulation) than on psy, pds, bkt; EBR50 treatment had a greater effect on the transcriptional expression of ipi-2, pds, lyc, crtR-B, bkt and crtO than on ipi-1 and psy. Furthermore, astaxanthin biosynthesis under EBR was up-regulated mainly by ipi1־ and psy at the post-transcriptional level, pds, lyc, crtR-B, bkt and crtO at the transcriptional level and ipi-2 at both levels.

Isolation and characterization of an Ashbya gossypii mutant for improved riboflavin production

The use of the filamentous fungus, Ashbya gossypii, to improve riboflavin production at an industrial scale is described in this paper. A riboflavin overproducing strain was isolated by ultraviolet irradiation. Ten minutes after spore suspensions of A. gossypii were irradiated by ultraviolet light, a survival rate of 5.5% spores was observed, with 10% of the surviving spores giving rise to riboflavin-overproducing mutants. At this time point, a stable mutant of the wild strain was isolated. Riboflavin production of the mutant was two fold higher than that of the wild strain in flask culture. When the mutant was growing on the optimized medium, maximum riboflavin production could reach 6.38 g/l. It has even greater promise to increase its riboflavin production through dynamic analysis of its growth phase parameters, and riboflavin production could reach 8.12 g/l with pH was adjusted to the range of 6.0-7.0 using KH2PO4 in the later growth phase. This mutant has the potential to be used for industrial scale riboflavin production.

Cultivation and identification of colon cancer stem cell-derived spheres from the Colo205 cell line

Our group established a method to culture spheres under serum-free culture condition. However, the biological characteristics and the tumorigenicity of spheres are unknown. Here, we demonstrate that sphere cells expressed high levels of the putative colorectal cancer stem cell markers CD133 and CD44. The CD133-positive rates were 13.27 ± 5.62, 52.71 ± 16.97 and 16.47 ± 2.45 percent in sphere cells, regular Colo205 cells and differentiated sphere cells, respectively, while the CD44-positive rates were 62.92 ± 8.38, 79.06 ± 12.10 and 47.80 ± 2.5 percent, respectively, and the CD133/CD44-double-positive rates were 10.77 ± 4.96, 46.89 ± 19.17 and 12.41 ± 2.27 percent, respectively (P < 0.05). Cancer sphere cells formed crypt-like structures in 3-D culture. Moreover, cells from cancer spheres exhibited more tumorigenicity than regular Colo205 cells in a xenograft assay. The cancer sphere cells displayed much higher oncogenicity than regular Colo205 cells to initiate neoplasms, as assayed by H&E staining, Musashi-1 staining and electron microscopy. Our findings indicated that the sphere cells were enriched with cancer stem cells (CSCs), and exhibited more proliferation capacity, more differentiation potential and especially more tumorigenicity than regular Colo205 cells in vitro and in vivo. Further isolation and characterization of these CSCs may provide new insights for novel therapeutic targets and prognostic markers.

Optimal subculture methods for passaging and growing human epidermal keratinocytes.

Background: Human epidermal keratinocytes (HEKs) cover the outer layer of the skin and play a key role in wound repair. Although the methods for isolation and cultivation of primary HEKs from epidermis have been used successfully in both laboratory and clinical settings, the ability to subculture (passage) these cells has yet to be established and is the primary factor hindering their usage. Objectives: We conducted this study to identify optimal subculture conditions for HEKs. Methods: We first harvested the primary HEKs from prepuce tissue specimens, and then compared three different reagent compositions (0.25% trypsin, 0.25% trypsin plus 0.01% EDTA, and 0.25% dispase digestion solution) for various periods of time at 4oC with the conventional 0.25% trypsin or 0.25% trypsin plus 0.01% EDTA digestion at room temperature. Results: Our data indicated that the cold digestion conditions yielded higher cell numbers and more viable cells than the conventional methods. Furthermore, the subcultured HEKs also adhered and grew better after four hours of a 0.25% trypsin cold digestion or after six hours of a 0.25% dispase cold digestion. These procedures produced higher numbers of HEK passages than that commonly seen experienced with conventional methods. Conclusion: The data from the current study demonstrated that the optimal subculture condition for passaging and growing HEKs in vitro is four hours digestion with 0.25% trypsin.