Pencil beam scanning dosimetry for large animal irradiation

The space radiation environment imposes increased dangers of exposure to ionizing radiation, particularly during a solar particle event. These events consist primarily of low-energy protons that produce a highly inhomogeneous depth–dose distribution. Here we describe a novel technique that uses pencil beam scanning at extended source-to-surface distances and range shifter (RS) to provide robust but easily modifiable delivery of simulated solar particle event radiation to large animals. Thorough characterization of spot profiles as a function of energy, distance and RS position is critical to accurate treatment planning. At 105 MeV, the spot sigma is 234 mm at 4800 mm from the isocentre when the RS is installed at the nozzle. With the energy increased to 220 MeV, the spot sigma is 66 mm. At a distance of 1200 mm from the isocentre, the Gaussian sigma is 68 mm and 23 mm at 105 MeV and 220 MeV, respectively, when the RS is located on the nozzle. At lower energies, the spot sigma exhibits large differences as a function of distance and RS position. Scan areas of 1400 mm (superior–inferior) by 940 mm (anterior–posterior) and 580 mm by 320 mm are achieved at the extended distances of 4800 mm and 1200 mm, respectively, with dose inhomogeneity <2%. To treat large animals with a more sophisticated dose distribution, spot size can be reduced by placing the RS closer than 70 mm to the surface of the animals, producing spot sigmas below 6 mm.     

蛇毒解聚素抑制荷U87胶质瘤裸鼠移植瘤的实验研究

目的 研究蛇毒解聚素在可移植性人脑胶质瘤间质化疗的疗效并探讨其作用机制.方法 建立BALB/c裸鼠U87胶质瘤移植瘤模型,将荷瘤裸鼠随机分为两组,采用间质内注射给药方法.蛇毒解聚素组:蛇毒解聚素40μg/d;对照组:为空白对照组,瘤内接种0.9%NaCl溶液,不给药,每组各15只裸小鼠.持续3周.定期观察肿瘤生长情况并测量肿瘤体积,绘制肿瘤生长曲线图并计算抑瘤率.全部BALB/c裸鼠移植瘤石蜡标本用SP法免疫组化染色,检测移植瘤组织中的微血管密度(MVD)及碱性成纤维细胞生长因子(bFGF)表达.结果 与对照组相比,蛇毒解聚素组均能抑制肿瘤生长(P＜0.05),其体积抑瘤率为50%,并能明显降低肿瘤MVD(14.16 4±2.49比38.01±6.07)和下调移植瘤组织中bFGF的蛋白表达(免疫反应积分为2.38±0.91比6.00±1.15).结论 蛇毒解聚索能明显抑制U87裸鼠移植瘤生长.     

莱菔硫烷抑制PMA诱导宫颈癌细胞迁移与侵袭

目的:观察莱菔硫烷(Sulforaphane,SFN)对宫颈癌细胞迁移和侵袭的抑制情况,并探讨其分子机制。方法:体外培养宫颈癌细胞系HeLa细胞,经100 nmol/L佛波脂PMA诱导24 h后,加入不同浓度的SFN继续孵育24 h。MTT法检测HeLa细胞的生长抑制情况;DCF染色法检测细胞内活性氧(Reactive oxygen species,ROS)的产生,细胞伤口愈合试验和小室侵袭实验分析SFN对PMA诱导HeLa细胞的迁移与侵袭情况。提取细胞总RNA,逆转录PCR检测基质金属蛋白酶-9(matrix met-alloproteinase-9,MMP-9)的表达,明胶酶谱实验检检测其酶活性。萤光素酶报告基因法检测SFN对核因子κB(Nuclear Fac-tor kappa B,NF-κB)活性的影响。结果:0～30μmol/L SFN对HeLa细胞的生长增殖无明显毒性作用。DCF染色显示PMA处理能使HeLa细胞ROS的含量增高5倍,而30μmol/L SFN处理能使ROS的产生降低44%;SFN也能使HeLa细胞迁移和侵袭率分别降低60%和75.5%。RT-PCR结果显示SFN能以剂量依赖性方式抑制PMA诱导的MMP-9表达,明胶酶谱实验显示MMP-9酶活性也明显降低。报告基因实验显示SFN能以剂量依赖性方式抑制NF-κB的活性。结论:SFN抑制NF-κB介导的MMP-9表达,从而影响宫颈癌细胞的迁移和侵袭。     

Loquat (Eriobotrya japonica) extracts suppress the adhesion, migration and invasion of human breast cancer cell line

We examined the inhibitory effects of loquat methanol extract on the adhesion, migration, invasion and matrix metalloproteinase (MMP) activities of MDA-MB-231 human breast cancer cell line. Cells were cultured with DMSO or with 10, 25, or 50 µg/ml of loquat methanol extract. Both leaf and seed extracts significantly inhibited growth of MDA-MB-231 cells in a dose-dependent manner, although leaf extract was more effective. Adhesion and migration were significantly inhibited by loquat extracts in a dose-dependent manner. Loquat extract also inhibited the invasion of breast cancer cells in a dose-dependent manner and leaf extract was more effective than seed extract. MMP-2 and MMP-9 activities were also inhibited by loquat extract. Our results indicate that methanol extracts of loquat inhibit the adhesion, migration and invasion of human breast cancer cells partially through the inhibition of MMP activity and leaf extract has more anti-metastatic effects in cell based assay than seed extract. Clinical application of loquat extract as a potent chemopreventive agent may be helpful in limiting breast cancer invasion and metastasis.     

Modeling the biological response of normal human cells,including repair processes,to fractionated carbon beam irradiation

To understand the biological response of normal cells to fractionated carbon beam irradiation, the effects of potentially lethal damage repair (PLDR) and sublethal damage repair (SLDR) were both taken into account in a linear-quadratic (LQ) model. The model was verified by the results of a fractionated cell survival experiment with normal human fibroblast cells. Cells were irradiated with 200-kV X-rays and monoenergetic carbon ion beams (290 MeV/u) at two irradiation depths, corresponding to linear energy transfers (LETs) of approximately 13 keV/μm and 75 keV/μm, respectively, at the Heavy Ion Medical Accelerator in Chiba of the National Institute of Radiological Sciences. When we only took into account the repair factor of PLDR, γ, which was derived from the delayed assay, the cell survival response to fractionated carbon ion irradiation was not fully explained in some cases. When both the effects of SLDR and PLDR were taken into account in the LQ model, the cell survival response was well reproduced. The model analysis suggested that PLDR occurs in any type of radiation. The γ factors ranged from 0.36–0.93. In addition, SLD was perfectly repaired during the fraction interval for the lower LET irradiations but remained at about 30% for the high-LET irradiation.     

金雀异黄素对胶质瘤细胞株细胞生长周期影响

目的 研究金雀异黄素(genistein)对人胶质瘤U251MG细胞生长和细胞周期的影响.方法采用噻唑蓝(MTT)比色法观察不同浓度genistein在不同作用时间下对U251MG细胞生长的影响,用流式细胞仪分析细胞周期分布,蛋白免疫印迹( western blotting)技术检测细胞周期素Bl(cyclinBl)和细胞周期素依赖性蛋白激酶1(CDK1)蛋白表达.结果Genistein对胶质瘤细胞生长有明显抑制作用,使细胞生长停滞于G2/M期,并使细胞cyclin B1、CDK1蛋白表达下降(均P＜0.01),且呈剂量依赖性.结论Genistein对U251 MG细胞生长有明显抑制作用,诱导G2/M期阻滞可能与下调cyclinB1和CDK1蛋白表达有关.     

胡桃醌抑制卵巢癌SKOV3细胞黏附和侵袭作用

目的探讨胡桃醌对人卵巢癌SKOV3细胞转移抑制作用。方法实验设对照组、胡桃醌12.5、25.0、50.0、100.0 μmol/L组,通过噻唑蓝(MTT)实验检测细胞增殖能力,黏附实验和Transwell实验观察胡桃醌对SKOV3细胞黏附和侵袭能力,Western blot 检测基质金属蛋白酶2(MMP-2)和MMP-9蛋白表达变化。结果与对照组比较,胡桃醌12.5、25.0、50.0、100.0 μmol/L组SKOV3细胞抑制率分别为(16.8±3.4)%、(39.6±9.7)%、(63.3±13.6)%和(84.5±17.3)%,明显升高,呈剂量依赖性;与对照组比较,25.0、50.0、100.0 μmol/L胡桃醌组SKOV3细胞黏附率和侵袭力分别为(49.2 ±6.5)%、(37.3±2.9)%、(24.7 ±3.7)%和(0.627±0.064)、(0.419±0.018)、(0.165±0.014),明显下降(P<0.05);与对照组比较,胡桃醌50.0、100.0 μmol/L组SKOV3细胞内MMP-2、MM-9表达水平明显下降(P<0.05),呈剂量依赖关系。结论胡桃醌能明显抑制SKOV3细胞黏附能力及侵袭力,其机制可能与抑制细胞内MMP-2、MMP-9表达有关。     

Capacity of proton beams in preserving normal liver tissue during proton beam therapy for hepatocellular carcinoma

Unirradiated liver volume (ULV) preservation rate is an important factor associated with radiation-induced liver disease (RILD) in patients with hepatocellular carcinoma (HCC) undergoing proton beam therapy (PBT). The purpose of this study is to identify the predictors for ULV preservation and quantify the capacity of proton beams in normal liver sparing during PBT. We reviewed planning data of 92 patients with single intrahepatic HCC tumors undergoing PBT. The potential clinical and planning factors that may affect ULV preservation were involved in multiple linear regression for ULV preservation rate. The significant factors were determined to be predictors and their influences were quantified. The median ULV preservation rate was 62.08%. All the assessed clinical factors showed significant effects on ULV preservation rate: clinical target volume (CTV), P < 0.001; portal vein tumor thrombosis (PVTT), P = 0.010; left lobe tumor, P = 0.010. In contrast, none of the planning factors demonstrated significance. The coefficients of significant factors in multiple linear regression were 60.85 for intercept, −0.02 for CTV, −9.01 for PVTT and 8.31 for left lobe tumors. The capacity of proton beams to spare normal liver tissue during PBT for HCC is mainly affected by clinical factors. The baseline of the ULV preservation rate is 60.85%, decreasing 0.02% with each milliliter of CTV increase and 9.01% for tumors with PVTT, and increasing 8.31% for tumors limited to the left lobe. Further clinical studies should be carried out to correlate our dosimetric findings with clinical outcomes.     

Long-term clinical outcomes of patients receiving proton beam therapy for caudate lobe hepatocellular carcinoma

Hepatocellular carcinoma (HCC) located in the caudate lobe (caudate HCC) is rare; however, patients with this type of tumour have poorer prognoses than those with HCC in other segments. Despite many published reports on the clinical usefulness of proton beam therapy (PBT) for HCC, data on the clinical outcomes of patients undergoing PBT for caudate HCC remain scarce. Therefore, the present study aimed to investigate the outcomes of this group of patients. Thirty patients with caudate HCC who underwent definitive PBT between February 2002 and February 2014 were retrospectively analysed. The total irradiation doses ranged from 55 to 77 (median 72.6) Gy relative biological dose. The median follow-up period was 37.5 (range, 3.0–152.0) months. The overall survival (OS) rates at one, three and five years were 86.6%, 62.8% and 46.1%, respectively. According to univariate and multivariate analyses, Child-Pugh A (P < 0.01), having a single tumour (P = 0.02) and a low serum alpha-fetoprotein level (AFP; P < 0.01) were significant factors predicting longer survival. The local control (LC) rates at one, three and five years were 100%, 85.9% and 85.9%, respectively, while the corresponding progression-free survival (PFS) rates were 65%, 27.5% and 22%, respectively. No grade 3 or worse adverse events were observed. PBT is effective and safe for the treatment of caudate HCC, and should therefore be considered a feasible option for intervention in patients with this disease.     

Inorganic sulfur reduces the motility and invasion of MDA-MB-231 human breast cancer cells

This study investigated the effects of inorganic sulfur on metastasis in MDA-MB-231 human breast cancer cells. MDA-MB-231 cells were cultured in the absence or presence of various concentrations (12.5, 25, or 50 µmol/L) of inorganic sulfur. Cell motility, invasion, and the activity and mRNA expression of matrix metalloproteases (MMPs) were examined. Numbers of viable MDA-MB-231 cells did not differ by inorganic sulfur treatment from 0 to 50 µmol/L within 48 h. Inorganic sulfur significantly decreased cell motility and invasion in the MDA-MB-231 cells in a dose-dependent manner (P < 0.05), as determined using a Boyden chamber assay and a Matrigel chamber. The activities of MMP-2 and MMP-9 were significantly reduced by inorganic sulfur in a dose-dependent manner (P < 0.05). The inorganic sulfur also significantly inhibited MMP-2 and MMP-9 expression in the cells (P < 0.05). These data suggest that inorganic sulfur can suppress cancer cell motility and invasion by inhibiting MMP-2 and MMP-9 activity and gene expression in MDA-MB-231 cells.     

Synthesis and biological evaluation in U87MG glioma cells of (ethynylthiophene)sulfonamido-based hydroxamates as matrix metalloproteinase inhibitors

Matrix metalloproteinases (MMPs) are important factors in gliomas since these enzymes facilitate invasion into the surrounding brain and participate in neovascularization. In particular, the gelatinases (MMP-2 and MMP-9), and more recently MMP-25, have been shown to be highly expressed in gliomas and have been associated with disease progression. Thus, inhibition of these MMPs may represent a promising non-cytotoxic approach to glioma treatment. We report herein the synthesis and biological evaluation of a series of 4-butylphenyl(ethynylthiophene)sulfonamido-based hydroxamates. Among the new compounds tested, a promising derivative, 5a, was identified, which exhibits nanomolar inhibition of MMP-2, MMP-9, and MMP-25, but weak inhibitory activity toward other members of the MMP family. This compound also exhibited anti-invasive activity of U87MG glioblastoma cells at nanomolar concentrations, without affecting cell viability.     

EGCG对人肝癌HepG2细胞增殖和侵袭的影响

目的探讨表没食子儿茶素没食子酸酯(EGCG)对肝癌HepG2细胞增殖和侵袭的影响及可能的分子机制。方法不同浓度EGCG对HepG2细胞进行干预后,CCK-8法检测EGCG对肝癌HepG2细胞增殖的影响;荧光显微镜及流式细胞术(FCM)Annexin V-FITC/PI双染法检测EGCG对HepG2细胞的凋亡诱导作用;Transwell侵袭实验检测EGCG对HepG2细胞侵袭的影响;ELISA法检测HepG2细胞中基质金属蛋白酶-2(MMP-2)和血管内皮生长因子(VEGF)的表达水平。结果EGCG各浓度组干预HepG2细胞24、48h后,细胞的生长增殖均明显受到抑制,其24h IC25值和IC50值分别为58.19和133.90mg/L。以60、135mg/L EGCG干预肝癌HepG2细胞24h后,FCM结果显示药物组细胞凋亡率明显高于对照组(P<0.05),荧光显微镜观察显示随着药物浓度的增加,细胞凋亡程度加重。Transwell侵袭实验显示,60、135mg/L EGCG对HepG2细胞侵袭力抑制率分别为69.47%和100%(P<0.05)。ELISA检测结果显示,EGCG干预后HepG2细胞上清液中MMP-2和VEGF表达水平下降(P<0.05)。结论 EGCG对人肝癌HepG2细胞具有抑制增殖和侵袭作用,其机制可能与EGCG诱导HepG2细胞凋亡和抑制肿瘤细胞中MMP-2及VEGF表达水平有关。     

High linear energy transfer carbon-ion irradiation upregulates PD-L1 expression more significantly than X-rays in human osteosarcoma U2OS cells

Programmed death ligand 1 (PD-L1) expression on the surface of cancer cells affects the efficacy of anti-PD-1/PD-L1 immune checkpoint therapy. However, the mechanism underlying PD-L1 expression in cancer cells is not fully understood, particularly after ionizing radiation (IR). Here, we examined the impact of high linear energy transfer (LET) carbon-ion irradiation on the expression of PD-L1 in human osteosarcoma U2OS cells. We found that the upregulation of PD-L1 expression after high LET carbon-ion irradiation was greater than that induced by X-rays at the same physical and relative biological effectiveness (RBE) dose, and that the upregulation of PD-L1 induced by high LET carbon-ion irradiation was predominantly dependent on ataxia telangiectasia and Rad3-related (ATR) kinase activity. Moreover, we showed that the downstream signaling, e.g. STAT1 phosphorylation and IRF1 expression, was upregulated to a greater extent after high LET carbon-ion irradiation than X-rays, and that IRF1 upregulation was also ATR dependent. Finally, to visualize PD-L1 molecules on the cell surface in 3D, we applied immunofluorescence-based super-resolution imaging. The three-dimensional structured illumination microscopy (3D-SIM) analyses revealed substantial increases in the number of presented PD-L1 molecules on the cell surface after high LET carbon-ion irradiation compared with X-ray irradiation.     

THSD7A在宫颈癌细胞侵袭、迁移等恶性生物学行为中的研究

目的 为了探索THSD7A对宫颈癌细胞的侵袭、转移以及凋亡等生物学行为的影响。方法 对宫颈癌C33a细胞和SiHa细胞培养及转染siTHSD7A基因,采用Western blotting和RT-qPCR方法在蛋白和基因水平对其验证。后应用Transwell小室实验、流式细胞术检测THSD7A沉默后对两种宫颈癌细胞的侵袭、转移、细胞周期及凋亡的改变。结果 与空白组和阴性对照组相比,沉默THSD7A的表达后,宫颈癌C33a细胞和SiHa细胞的侵袭、迁移能力明显下降（P＜0.01）;进入S期和G2/M期细胞数目减少,凋亡比例增高（P＜0.05）。结论 提示THSD7A可能作为一种致癌基因,在宫颈癌恶性生物学行为的发展过程中起到推进作用。     

MK-siRNA对人乳腺癌细胞的生长、粘附及迁移能力的影响

目的:探讨siRNA MK转染对乳腺癌MCF-7细胞增殖和转移能力的影响。方法:构建特异性抑制Midkine基因siRNA片段,转染至乳腺癌MCF-7细胞中,CCK-8法比较细胞增殖能力的改变,体外迁移实验比较细胞运动能力的改变,基质胶粘附实验比较细胞粘附率的变化。结果:MK表达下调后,MCF-7细胞的增殖能力明显降低(P<0.05),体外迁移能力显著降低(P<0.05),siRNA细胞对基质胶的粘附率明显降低(P<0.05)。结论:MK基因表达下调能降低乳腺癌细胞迁移运动和降低细胞对基质胶的粘附率,并降低肿瘤细胞的增殖,提示MK在乳腺癌恶性进展中起重要作用。     

木犀草素对脑胶质瘤细胞氨肽酶N活性影响

目的探讨木犀草素对脑胶质瘤细胞氨肽酶N活性影响机制。方法本研究采用酶抑制动力学方法, 研究了木犀草素对氨肽酶N的抑制作用、抑制类型和抑制动力学常数, 并进行了脑胶质瘤U87细胞的凋亡生长抑制试验。结果木犀草素能够诱导了脑胶质瘤U87细胞的凋亡, 并呈现一定的剂量依赖性, 当浓度≥10 μmol时, 与阳性对照bestatin 相比差异有统计意义(P<0.05), 求得半细胞凋亡率IC₅₀为(61.23±2.13)μmol;此外木犀草素是一种可逆的竞争型氨肽酶N抑制剂, 半抑制率IC₅₀为(70.85± 2.05)μmol, 抑制常数Ki为(24.83± 0.14)μmol;失活动力学时间进程分析表明,木犀草素能快速与氨肽酶N发生作用并迅速降低酶的活性。结论木犀草素是一个竞争性的氨肽酶N抑制剂, 使氨肽酶N的活性降低, 对诱导了脑胶质瘤U87细胞的凋亡起到重要作用。     

X射线对人肝癌血管内皮细胞迁移的影响及其机制

目的 探讨X射线对人肝癌血管内皮细胞（TEC）迁移的影响并探讨其机制。方法 细胞划痕实验检测TEC细胞迁移能力，实时荧光PCR法检测Plk1 mRNA表达水平，免疫印迹法检测Plk1、STAT3及磷酸化STAT3（p-STAT3Y705）水平。结果 2 Gy X射线照射后24 h TEC迁移距离为（114.37±35.12）像素，对比未受照射TEC迁移距离（78.89±24.67）显著升高（P<0.01）；2 Gy X射线照射后Plk1 mRNA表达水平显著升高（P<0.05），BI2536抑制Plk1激酶活性后p-STAT3Y705水平降低；2 Gy X射线照射后Plk1和p-STAT3Y705水平显著升高。结论 2 Gy X射线通过Plk1磷酸化STAT3（而非增加STAT3表达水平）来发挥其促进TEC迁移作用。     

双氢青蒿素抑制胶质瘤细胞生长机制

目的 探讨双氢青蒿素对胶质瘤细胞C6生长影响及可能机制。方法 培养大鼠脑胶质瘤C6细胞,噻唑蓝法检测双氢青蒿素对C6细胞生长抑制率,annexin V/PI检测C6细胞凋亡率;ELISA法检测细胞培养液上清中cyclinD1、Bax和casepase-3蛋白表达变化;Western blot法检测Bax和Bcl-2蛋白表达量。结果 双氢青蒿素可明显抑制C6细胞生长,以作用48 h最为明显,0.05、0.50、5.00和50.00μmol/L双氢青蒿素组细胞生长抑制率分别为(83.96±1.08)% 、(74.04±3.54)% 、(68.70±9.20)% 和(57.25±7.01)%,与对照组(100.00±1.07)% 比较,差异有统计学意义(P<0.01);50.00μmol/L双氢青蒿素组细胞凋亡率[(25.98±3.95)%]明显高于对照组[(2.32±0.78)%],差异有统计学意义(P<0.01);与对照组比较,各剂量双氢青蒿素组C6细胞培养液上清中caspase-3分泌增加,50.00μmol/L双氢青蒿素组细胞Bax蛋白表达增加,Bcl-2蛋白表达减少,Bax/Bcl-2比值增加。结论 双氢青蒿素通过下调cyclin D1蛋白,上调Bax/Bcl-2比值使caspase-3蛋白活化,从而抑制胶质瘤细胞生长。     

异甘草素对小儿脑胶质瘤细胞增殖、凋亡的影响及相关机制研究

目的 研究异甘草素对脑胶质瘤U251细胞增殖、凋亡的影响,并探讨其相关分子机制.方法 取对数生长期人脑胶质瘤U251细胞,用不同浓度(0、10、20、40、60、80μmol/L)异甘草素处理,每个浓度设5个复孔.异甘草素处理24h、48h、72h后,采用四甲基偶氮唑蓝(MTT)法检测细胞增殖能力,采用流式细胞术检测细胞凋亡的变化,逆转录-聚合酶链反应(RT-PCR)法检测FoxM1 mRNA的表达水平.结果 U251细胞经过浓度为0、10、20、40、60、80μmol/L的异甘草素处理24h、48h及72h后,细胞增殖均不同程度地受到抑制.在相同作用时间,不同浓度之间细胞增殖情况比较差异有统计学意义(F值分别为38.5、98.9、108.1,均P＜0.05);在相同异甘草素浓度,不同作用时间(24h、48h、72h)细胞增殖情况比较差异均有统计学意义(F值分别为11.4、20.8、43.1、53.9、30.4,均P＜0.05).不同浓度异甘草素作用于U251细胞24h后,随着异甘草素浓度的增加,细胞凋亡率增加(F=120.3,P＜0.05).不同浓度异甘草素处理U251细胞48h后,FoxM1 mRNA相对表达量分别为(71.2±5.1)％、(54.6±3.3)％、(35.4±4.1)％、(20.8±4.0)％,比较差异有统计学意义(F=83.5,P＜0.05).结论 异甘草素体外可有效抑制U251细胞的增殖并诱导其凋亡,其分子机制可能与下FoxM1基因的表达有关.     

恶性人脑胶质瘤的HSV-Tk/GCV自杀基因治疗系统的构建及应用研究

目的构建恶性人脑胶质瘤细胞株TJ899的单纯疱疹病毒的胸腺激酶/丙氧鸟苷（HSV-Tk/GCV）自杀基因治疗系统，探讨该体系在脑胶质瘤治疗中的作用。方法采用PCR、RT-PCR、融合PCR、酶切、连接等技术构建由自杀基因pcDNA3.1（-）CMVTK表达载体；用脂质体介导法将pCMVTK质粒转染进入TJ899恶性人脑胶质瘤细胞，并用M IT法检测感染后细胞对（GCV）的敏感性。结果pCMVTK质粒经测序证实含有TK目的基因；脂质体介导Pcmvtk质粒成功转染了TJ899细胞，并在体外实验研究中显示很好的治疗效果。结论建立了作用于恶性人脑胶质瘤细胞株TJ899的HSV-Tk/GCV自杀基因治疗系统，为脑胶质瘤的临床研究打下了基础。