低频低强度超声对巨噬细胞内耻垢分枝杆菌的损伤效应

目的 探讨低频低强度超声(low frequency low intensity ultrasound,LFLIU)对巨噬细胞内耻垢分枝杆菌的损伤效应及机制.方法 将巨噬细胞RAW264.7分为超声辐照0、5、10 min和15 min组(n=3),应用MTr比色法分析不同超声强度对RAW264.7细胞存活率的影响,采用流式细胞术检测RAW264.7细胞经超声辐照以及吞噬细菌后的凋亡率和坏死率,使用平板计数法评估RAW264.7细胞内耻垢分枝杆菌活性,使用激光共聚焦显微镜观察RAW264.7细胞经超声辐照后活性氧(reactive oxygen species,ROS)的产生.结果 超声辐照声强为0.14 W/cm2,辐照时间为5、10 min时,RAW264.7细胞活性未见明显影响,存活率分别为(116.37±9.54)％、(97.14±9.26)％;经0.14W/cm2超声辐照5 min后,RAW264.7细胞的凋亡率和坏死率显著大于超声辐照0 min组(P＜0.05);平板菌落计数结果显示,RAW264.7细胞内耻垢分枝杆菌经声强0.14 W/cm2超声辐照5、10 min后活性均低于超声辐照0 rain组(P＜0.05);激光共聚焦观察到0.14 W/cm2超声刺激RAW264.7细胞产生了ROS.结论 LFLIU能诱导RAW264.7细胞凋亡和产生ROS,并导致RAW264.7细胞内耻垢分枝杆菌活性下降.     

超声辐照对主动脉平滑肌细胞增殖的抑制作用

目的　观察不同剂量的超声辐照对牛主动脉平滑肌细胞 (CASMC)增殖的影响 ,寻找出抑制细胞增殖的最佳辐照剂量 ,为临床治疗再狭窄提供理论依据。方法　体外培养的牛主动脉平滑肌细胞传至 3～ 8代 ,分为 4组 :第 1组为对照组 ;第 2组为超声辐照组 ;第 3组为血管紧张素Ⅱ (ATⅡ )组 ;第 4组为超声辐照+ATⅡ组。在 2 4孔和 96孔培养板上分别进行超声辐照 ,用细胞计数法、MTT法、3 H -TdR掺入法协同检测细胞增殖程度 ,采用多因素分析法逐步寻找出超声抑制细胞增殖的最佳辐照剂量。结果　 2 .2MHz,0 .5W /cm2 ,6 0s的超声剂量可以抑制CASMC增殖 ,抑制率达到 2 0 %～ 30 % (P <0 .0 1)。随着声强提高 ,辐照时间延长 ,对细胞的抑制率亦增高。在相同频率下 ,作用能量达到 1.0W /cm2 ,6 0s时 ,对细胞开始产生杀伤作用。结论　一定频率和剂量的超声辐照可以抑制CASMC增殖。     

Inhibitory effect of ultrasound microbubbles carrying HSV1-TK gene combined with ganciclovir on prostate cancer cells

目的 观察超声微泡造影剂携单纯疱疹病毒Ⅰ型胸昔激酶(HSV1-TK)基因体外转染及联合前药更昔洛韦(GCV)对前列腺癌细胞株PC-3的抑制效应.方法 超声辐照PC-3细胞,MTT筛选出最适辐照时间;将含有HSV1 -TK基因的质粒通过静电吸附在脂质微泡表面,采用最适超声辐照转染,并用荧光显微镜及Western blot观察TK基因的转入及表达;MTT法观察使用不同浓度的前药更昔洛韦(GCV)后细胞增殖抑制率;流式细胞仪检测最适浓度的前药GCV对转染后的前列腺癌细胞PC-3的周期的影响;MTT法观察HSV1-TK/GCV自杀基因系统对前列腺癌PC-3细胞杀伤效应.结果 MTT检测显示超声强度为1.0 W/cm2,频率为1 MHz,辐照时间30 s时超声辐照对细胞无明显的抑制作用;通过超声辐照后HSV1-TK基因可以顺利转入PC-3细胞中并有稳定的表达.与对照组相比,当前药GCV浓度在100μg/ml时,超声+微泡+TK组细胞杀伤率明显高于其他各组,细胞存活率约32％,低于其他各组(P＜0.05);流式细胞术检测显示大多数细胞被阻断在S期,细胞生长受到明显抑制.结论 以超声辐照微泡造影剂为载体转染自杀基因联合前约GCV,对人前列腺癌细胞有明显杀伤作用.     

携HSV1-TK自杀基因超声微泡造影剂联合前药更昔洛韦对前列腺癌细胞的抑制

目的观察超声微泡造影剂携单纯疱疹病毒Ⅰ型胸昔激酶(HSV1-TK)基因体外转染及联合前药更昔洛韦(GCV)对前列腺癌细胞株PC-3的抑制效应。方法超声辐照PC-3细胞,MTT筛选出最适辐照时间;将含有HSV1-TK基因的质粒通过静电吸附在脂质微泡表面,采用最适超声辐照转染,并用荧光显微镜及Western blot观察TK基因的转入及表达;MTT法观察使用不同浓度的前药更昔洛韦(GCV)后细胞增殖抑制率;流式细胞仪检测最适浓度的前药GCV对转染后的前列腺癌细胞PC-3的周期的影响;MTT法观察HSV1-TK/GCV自杀基因系统对前列腺癌PC-3细胞杀伤效应。结果 MTT检测显示超声强度为1.0 W/cm2,频率为1 MHz,辐照时间30 s时超声辐照对细胞无明显的抑制作用;通过超声辐照后HSV1-TK基因可以顺利转入PC-3细胞中并有稳定的表达。与对照组相比,当前药GCV浓度在100μg/ml时,超声+微泡+TK组细胞杀伤率明显高于其他各组,细胞存活率约32%,低于其他各组(P<0.05);流式细胞术检测显示大多数细胞被阻断在S期,细胞生长受到明显抑制。结论以超声辐照微泡造影剂为载体转染自杀基因联合前药GCV,对人前列腺癌细胞有明显杀伤作用。     

微泡造影剂结合超声介导一氧化氮合成酶基因转染血管平滑肌细胞

目的 探讨微泡造影剂及超声辐照介导内皮型一氧化氮合成酶(eNOS)基因转染体外培养大鼠血管平滑肌细胞(VSMCs)的可行性.方法 实验分为A、B、C、D、E5组,分别为空白对照组、单纯质粒浸泡组、质粒+微泡造影剂组、质粒+超声辐照组、质粒+超声辐照+造影剂组.用重组真核表达载体pcDNA3.1- eNOS经微泡造影剂及超声辐照介导转染体外培养的大鼠VSMCs,辐照条件为超声探头频率为10 MHz,机械指数为1.9,辐照时间为10 min,重组真核细胞表达载体pcDNA3.1-eNOS 5μg/mL.辐照48 h后,采用RT-PCR、Western blot及免疫组化检测VSMCs内eNOSmRNA和蛋白的表达.结果 RT -PCR检测E组eNOS mRNA的表达最显著,积分吸光度(IA)比值为(91.11±3.41)％,B、C、D组也有少量表达,IA比值分别为(26.10±1.32)％、(31.42±2.43)％、(35.05±2.25)％,与E组相比均P＜0.05.蛋白印迹分析eNOS蛋白表达,A组有极少量表达,B、C、D组也有少量表达,IA比值分别为(22.12±1.33)％、(25.42±2.41)％、(33.11±3.11)％,而E组表达最明显,IA比值为(84.22±9.22)％,与各组相比均P＜0.05.结论 超声辐照结合微泡造影剂能显著提高eNOS基因在VSMCs的转染效率,提高细胞内一氧化氮合成酶的表达.     

低强度超声联合5-氨基酮戊酸对大鼠骨肉瘤UMR-106细胞的杀伤机制研究

目的：研究低强度超声结合5-氨基酮戊酸（5-ALA）对大鼠骨肉瘤UMR-106细胞的杀伤机制。方法将处于对数生长期的大鼠骨肉瘤UMR-106细胞分成对照组、5-ALA组、超声组和超声＋5-氨基酮戊酸组（SDT组）。流式细胞仪检测细胞凋亡率,活性氧（ROS）的产生,线粒体膜电位（MMP）的改变;荧光显微镜观察33342染色细胞核的形态改变;透射电镜观察超微结构改变。结果当超声频率为1．0MHz,声强2．0W/cm2,5-ALA浓度2mmol/L,SDT组与对照组、超声组及5-ALA组比较,其凋亡率（32．2±1．4）％,明显增高（P＜0．05）。同时伴有ROS（34．4±2．4）％的产生和MMP（42．2±2．6）％的降低。通过33342染色荧光显微镜观察到超声联合5-ALA组的细胞核发生浓缩和碎裂。透射电镜观察到细胞膜、线粒体、高尔基体等细胞器改变及凋亡小体的形成。结论低强度超声联合5-ALA对UMR-106细胞的杀伤作用明显,细胞以凋亡为主,其线粒体途径对UMR-106细胞凋亡起着重要作用。     

低强度脉冲超声波促进皮肤成纤维细胞增殖的实验研究

目的:研究不同辐照参数(频率、声强、辐照时间)低强度脉冲超声波(LIPUS)对成纤维细胞增殖的影响.方法:采用MTT法测定频率1MHz、1.2MHz、1.7MHz,每一频率下四种声强的LIPUS,同一声强分别辐照5min、10min、15min、20min和30min体外培养的人真皮成纤维细胞24h后的生长增殖情况,筛选出促进成纤维细胞生长的最适宜频率、声强和辐照时间.结果:①在MTT法测定细胞活性中三种频率(1MHz、1.2MHz、1.7MHz),每一频率下四种声强的LIPUS辐照成纤维细胞5min,24h后吸光光度值均增加,辐照成纤维细胞10min,24h后吸光光度值增加最高,较对照组都有显著性差异(P＜0.01).超过一定辐照时间后,吸光光度值下降,且随着辐照时间的延长,吸光光度值下降显著.②频率1MHz、声强0.1W/cm2、辐照时间10min下的细胞相对成活率最高,与对照组比较差异有统计学意义(P＜0.01).结论:不同频率、不同声强的LIPUS辐照一定时间均能促进人真皮成纤维细胞增殖,超过一定辐照时间则会抑制细胞生长.而频率1MHz、声强0.1W/cm2、辐照时间10min为促进成纤维细胞增殖较为适宜的辐照参数.     

聚焦超声诱导卵巢癌细胞株COC1凋亡及其机制的初步研究

目的:研究低频脉冲聚焦超声波对人卵巢癌细胞株COC1的抗肿瘤作用,并探讨其诱导细胞凋亡的分子机制.方法:以低频脉冲聚焦超声(超声频率0.8MHz,声强83.6W/cm2,脉肿持续时间:5μs)辐照卵巢癌COC1细胞10s,然后采用MTT法观察超声对COC1细胞增殖的抑制作用;流式细胞仪(FCM)、TUNEL法、荧光染色和光、电镜检测细胞凋亡;应用FCM免疫荧光技术观察Bcl-2、bax基因蛋白表达的变化.结果:超声可明显抑制存活卯巢癌COC1细胞的增殖,辐照后12h、24h、36h、48h,COC1细胞MTT光吸收值与对照组相比显著降低;FCM检测超声辐照后6h的COC1细胞凋亡率达(23.57±4.62)%;Bcl-2蛋白的荧光指数(FI)降低,而bax蛋白FI升高.同时TUNEL法观察到棕褐色着染的凋亡细胞;荧光显微镜和电镜下观察到凋亡小体形成等典型的形态学改变.结论:低频脉冲聚焦超声辐照对人卵巢癌细胞株COC1具有细胞毒作用,其机制为诱导辐照后存活的人卵巢癌细胞株COC1凋亡,bcl-2和bax蛋白可能参与了COC1细胞凋亡过程.     

超声造影剂对血管平滑肌细胞增殖的影响

目的观察超声造影剂对大鼠胸主动脉血管平滑肌细胞(VSMC)增殖的影响.方法血小板衍生生长因子-BB(PDGF-BB)刺激大鼠VSMC建立细胞增殖模型.在培养基中加或不加脂质体微泡(1μl/ml),采用台盼蓝排斥染色、MTT法和3H-TdR掺入法检测不同强度、时间的连续波超声辐照对VSMC的毒性作用以及增殖和DNA合成的抑制作用.结果频率1MHz、声强0.3W/cm2的超声联合造影荆辐照VSMC,未见细胞溶解,24h后细胞核蓝染率较辐照即刻显著降低(P＜0.01);辐照30s即可显著抑制PDGF-BB所致的VSMC增殖和DNA合成(P＜0.05),随着辐照时间延长,抑制作用增强,60s时抑制程度最强(P＜0.01).结论低强度超声辐照下超声造影荆的空化效应可抑制VSMC增殖,并对细胞膜有暂时性的损伤作用.     

携自杀基因微泡转染卵巢癌细胞株SKOV_3的超声参数及转染效率

目的:探究超声介导携Ⅰ型单纯疱疹病毒胸苷激酶(hsv1tk)自杀基因微泡转染SKOV3细胞的超声参数及转染效率.方法:超声在不同微泡浓度与辐照时间(8,15,30,60s间隔1s)组合下辐照SKOV3细胞,MTT法筛选最适辐照时间和微泡浓度.将实验分成6组分别进行以下处理:超声辐照组,脂质体+超声辐照组,脂质体+微泡+超声辐照组,微泡+超声辐照组,裸质粒组(阴性对照),脂质体组(阳性对照).用裸质粒及脂质体处理后分别转染pcDNA3.1-EGFP/hsv1tk质粒;采用荧光显微镜、流式细胞技术分别定性和定量检测各组转染效率;采用逆转录聚合酶链反应(RT-PCR)方法检测hsv1tk基因的表达.结果:MTT检测在超声强度0.5W/cm2,频率1MHz,微泡浓度0.56×1011个/L,辐照时间8s间隔1s条件下,超声辐照微泡转染对细胞活性无明显抑制.经此条件转染后荧光显微镜下可观察到脂质体+微泡+超声辐照组的绿色荧光强度最强;流式细胞技术检测表明,微泡+超声辐照组转染效率为(11.74±0.19)%,比超声辐照组(2.19±0.22)%高,而脂质体+微泡+超声辐照组(25.62±0.08)%均高于其他各组(P<...     

Value of D-Dimers in patients with acute aortic dissection

Objective: To evaluatel the value of D-dimers in patients with acute aortic dissection (AAD). Methods: This study consisted of 16 patients with AAD and 27 non-AAD patients. Serum D-dimets were measured by Sta-Liatest D-DI immunoturbidimetric assay. Results: D-dimer level was higher (P ＜ 0.001) in patients with AAD(7.91 ± 5.52 μg/ml) than that in non- AAD group(1.57±1.24 μg/ml). D-dimer was positive (＞0.4 μg/ml) in all patients with AAD and in 10 control group patients (37%). Among patients with acute AAD, D-dimers tended to be higher in Stanford A than in Stanford B (8.67 ± 4.31 μg/ml vs. 3.24±1.27 μg/ml, P ＜0.01). D-dimer values tended to be higher in more extended disease(3.84 ± 1.65 μg/ml, 8.57 ± 3.58 μg/ml and 11.87 ± 5.69 μg/ml in thoracic aorta, thoracic and abdominal aorta, thoracic and abdominal aorta and iliacal arteries, respectively, P ＜ 0.05 for both 8.57 ± 3.58 and 11.87 ± 5.69 vs. 3.84 ± 1.65 ). Including the control group into the analysis, we found a sensitivity of 100%, a negative predictive value of 100%, and a specificity of 66% and a positive predictive value of 64% for D-dimer in diagnosis of AAD in our patients with suspected AAD. Conclusion: D-dimer was elevated in patients with AAD. A negative D-dimer test result could be useful in excluding AAD.     

Research of combined liver-kidney transplantation model in rats

Objective： To set up a simple and reliable rat model of combined liver-kidney transplantation. Methods： SD rats served as both donors and recipients. 4℃ sodium lactate Ringer＇s was infused from portal veins to donated livers,and from abdominal aorta to donated kidneys, respectively. Anastomosis of the portal vein and the inferior vena cava （IVC） inferior to the right kidney between the graft and the recipient was performed by a double cuff method, then the superior hepatic vena cava with suture. A patch of donated renal artery was anastomosed to the recipient abdominal aorta. The urethra and bile duct were reconstructed with a simple inside bracket. Results： Among 65 cases of combined liver-kidney transplantation, the success rate in the late 40 cases was 77.5%. The function of the grafted liver and kidney remained normal. Conclusion： This rat model of combined liver-kidney transplantation can be established in common laboratory conditions with high success rate and meet the needs of renal transplantation experiment.     

BLOOD PRESSURE CHANGE WITH AGE IN SALT-SENSITIVE TEENAGERS

Objective To observe blood pressure change with age in salt-sensitive teenagers whose salt sensitivity were determined by repeated testing.Methods Salt sensitivity was determined through intravenous infusion of normal saline combined with volume-depletion by oral diuretic furosemide in 55 teenagers. After five years, salt sensitivity was re-examined and subject blood pressure was followed up. Blood pressure changes in salt-sensitive teenagers were compared to that of non-salt sensitive teenagers over five years.Results After 5 years, the repetition rate of salt sensitivity determined by intravenous saline loading is 92.7%. In teenagers with salt sensitivity on the baseline, both the systolic blood pressure increments and increment rates were much higher than non-salt sensitive teenagers (12.7±12.1 mmHg vs. 2.8±5.2 mmHg, P＜ 0.01; 12.2%± 12.0% vs. 2.5% ±4.4%, P＜ 0.001,respectively). There was a similar trend for diastolic blood pressure (8.4 ± 6.4 mmHg vs. 3.7 ± 6.4 mmHg, P = 0.052; 13.2% ±10.6 % vs. 6.8%± 10.1%, P = 0.053, respectively).Conclusions Salt sensitivity determined by intravenous saline loading showed good reproducibility. Blood pressure increments with age were much higher in salt-sensitive teenagers than non-salt sensitive teenagers, especially in terms of systolic blood pressure.     

安心颗粒对急诊经皮冠状动脉介入术后生活质量影响的研究

目的：评价使用安心颗粒对急诊经皮冠状动脉介入术（PPCI）术后生活质量的影响.方法：将160例接受PPCI的急性ST段抬高型心肌梗死患者随机分为安心颗粒组（术前顿服安心颗粒8.8g,术后安心颗粒4.4 g/次,每日2次）和对照组（仅接受基础药物治疗）.所有患者均服用阿司匹林、氯吡格雷和阿托伐他汀.分别在入院时、出院前1d、出院后180 d时,应用心肌梗死多维度量表（MIDAS）、中文版SF-36评价量表对患者生活质量评分.并观察术后30 d以内的出血并发症、血小板减少症发生情况.结果：入院时和出院前1d,两组患者的心肌梗死MIDAS、SF-36量表评分比较无差异（P＞0.05）;出院后180 d时,与对照组比较,安心颗粒组MIDAS、SF-36评分明显减低（P＜0.05）;组内与入院时比较,两组出院前1d、出院后180 d时,MIDAS、SF-36评分均降低（P＜0.05）.两组患者在随访期间均无大量出血、少量出血、重度和极重度血小板减少症发生,安心颗粒组有4例、对照组有7例发生不明显出血（P＞0.05）.两组发生轻度血小板减少症的患者数比较无差异（P＞0.05）.结论：PPCI使用安心颗粒,能改善急性ST段抬高型心肌梗死患者的生活质量,且不增加出血风险.     

The comparative studies of the influences of Urapidil and Nicardipine on sino-atrial node function, atrio-ventricular node function and hemodynamics

Objective:To investigate the influences of urapidil and nicardipine on rabbit sinus function,atrio-ventricular node function and hemodynamics.Methods:Thirty-two Angora's rabbits were selected and randomly divided into four groups.U1 group:urapidil 0.25 mg/kg;U2 group:urapidil 0.5 mg/kg;N1 group:nicardipine 10 μg/kg;N2 group:nicardipine 20 μg/kg.All these medicine were administrated within 30 seconds.Measurements were taken before and after the administration of urapidil or nicardipine for the following data:mean blood pressure(MAP),heart rate(HR),sino-atrial conduction time(SACT),maximal sinoatrial recovery time(SNRTmax)corrected sinus node recovery time(CSNRT),index of sinus node recovery time(SNRTI),Wenckebach A-V conduction frequency (WB),and P-R interval.Results:Significant MAP and HR changes were identified in all of the four groups before and after administration of both urapidil and nicardipine.No significant changes could be found in the rest of the parameters.Intergroup analysis showed that SACT and CSNRT of N1 and N2 groups were shorter than those of the U2 group(P＜0.01);the MAP decreased(P＜0.01)and the HR increased drastically(P＜0.01).Conclusions:Neither urapidil(0.25 mg/kg,0.5 mg/kg)nor nicardipine(10μg/kg,20μg/kg)has any significant influence on rabbit sinus function or rabbit atrio-ventricular node function.Nicardipine could be a better choice than urapidil for parafunctional sinus node patients.     

Expression of OPGmRNA and ODFmRNA in the patients of hip fracture due to osteoporosis

Objective：To investigate the gene expression of osteoprotegerin（OPG） and osteoclast differentiation factor（ODF） in the bone tissue of patients with hip fracture due to osteoporosis. Methods：OPGmRNA and ODFmRNA in the bone tissue in 50 cases of osteoporosis sufferers（over 50 years old） with hip fracture（Observer Group） and 30 cases of hip facture sufferers with no osteoporosis（Control group） were analyzed with the Semi-Quantitative RT-PCR method. Results：The mRNA expressed of ODF, OPG were both high in the patients with hip fracture. In the control group, the expression of OPG mRNA was observed, while the expression of ODF mRNA was very slight. Conclusion：Aged patients contained all signals including OPG, ODF that are essential for inducing osteoclastogenesis and promoting bone resorption.     

Dynamic Changes in Serum Estradiol and Progesterone levels in Patients of Premenstrual Syndrome with Adverse Flow of Liver-qi

Objective:To probe into the influence of changes of ovarian hormones on the pathogenesis of the specific sub-type premenstrual syndrome(PMS)and reveal partial microcosmic mechanisms of adverse flow of liver-qi.Methods:Estradiol(E2)and progesterone(P)levels in serum were determined at different phases of menstrual cycle by radioimmunoassay.Results:In the group of PMS with adverse flow of liver-qi.the secretive peak value Of E2 and P at the follicular phase significantly decreased,and the secretive peak value at the luteal phase did not come into being.Conclusions:Low E2 and P secretive peak at the follicular phase and absence of secretive peak at the luteal phase is one of the microcosmic mechanisms of PMS with adverse flow of liver-qi.One of the pathophysiologic mechanisms of specific sub-type PMS is probably the continuous low level of E2and P.     

Real-time Three-dimensional Echocardiography in Assessment of Left Ventricular and Right Ventricular Volumes

Real-time three-dimensional echocardiography (RT3DE)is a new ultrasound technique that enables dynamic threedimensional visualization and quantification of the heart in real time. Investigation of feasibility and methodology of RT3DE in determining left ventricular (LV) and right ventricular (RV) volumes, RT3DE was performed in 35 normal adults using Philips SONOS 7500 system with a 2-4 MHz matrix array transducer. The 60°×60° "pyramid" volume database was obtained and analyzed on a TomTec echo workstation. Both LV and RV volumes were calculated with four 3DE methods (i.e. apical 2, 4, 8, and 16-plane) through manually tracing ventricular endocardial borders in end diastole and end systole. Stroke volumes were then calculated. LV volume was also measured by 2DE Simpson's rule using GE VIVID 7 ultrasound machine.     

SCREENING FOR A 21-CHROMOSOME ABNORMALITY IN PREIMPLANTED EMBRYOS OF ELDERLY WOMEN

Increasing maternal age is the only etiological factor unequivocally linked to Down's syndrome in humans. The occurrence rate of newborns with Down's syndrome is about 1/220 in women over 35 years old. However, the occurrence rate in embryos fertilized in vitro, of the elder woman is unclear. Using FISH we screened the number of chromosome 21 in preimplanted embryos of 5 elderly women (average age, 38.4 years) to study the feasibility and necessity of screening trisomy 21 in embryos in patients over 35 years old at the in vitro fertilization (IVF) center.     

Scientific principles and rigorous processes should be followed in developing clinical guidelines for therapeutic interventions of integrative medicine

A clinical guideline for the therapeutic interventions of integrative medicine may be defined as a written document which states a series of recommendations on therapeutic interventions of integrative medicine for a special disease or condition. The guideline may provide assistance to medical professionals in making clinical decisions aimed at improving the clinical outcome of patients and reducing the costs of medical care（~＇4~. Recommendations issued by a guideline should be based on the best available evidence in both Western and Chinese medicine. For fulfilling this purpose, the development of clinical guidelines for therapeutic interventions in the field of integrative medicine should follow scientific principles and undergo a rigorous processes.