1 Introduction

Over the last 20 years,the global incidence of malignant tumors has increased steadily[1].The current clinical experience has revealed signi ficant differences in radiosensitivity among different types of tumors[2,3],but also differencesinradiosensitivityamongdifferentindividualswith thesametumortype[4–7].Thismeansthatbasicresearchon tumor radiotherapy is very important,as it provides an experimental basis and theoretical support for the personalizedtreatmentofpatientstoincreasetheprecisionofspeci fic radiotherapy treatments.For the radiosensitivity of tumor cells,theradiationsourceisusedtoirradiatecells,sothatthe cells receive a certain dose,so as to observe its radiosensitivity.Irradiation conditions are different for each research unit.In recent years,irradiation with a 6 MV X-ray linear accelerator has been conducted in most hospitals by using different irradiation methods.Yang et al.[8].used 6 MV X-ray irradiation treatment on MDA-MB-231 cells during the logarithmic growth phase;the source-to-skin distance(SSD)was noted to be 100 cm,but the build-up and backscattering materials were not stated.

Some studies have described the irradiation conditions and methods;however,the description was not suf ficiently detailed.A study by Jin et al.[9].described cells that were irradiated with 6 MV X-ray radiation with an SSD of 100 cm and an irradiation field of 25×25 cm2.The buildup and back-scattering materials were not explained.In a study by Chen et al.[10],a vertical beam irradiation method with 6 MV X-ray radiation,gantry angle of 180°,and 1.5 cm of build-up materials were adopted whereas Zachari et al.[11].irradiated cells with 6 MV X-ray radiation and a 10×10 cm2irradiation field.In summary,many studies on the radiosensitivity of various tumors have been reported;however,the speci fic irradiation conditions and methods differ between studies,which mean that many irradiation experiments cannot be reproduced between different research centers.Meanwhile,it has become ever more important to establish a convenient and feasible cell irradiation method able to deliver an accurate dose.

Thequestionofwhetherthecellreceivesthepresetdoseis of importance.Without other factors,and only considering thesource-celldistance(SCD),build-upandback-scattering effect,cavity effect,and the convenience and feasibility of cell culture,three cell irradiation methods were designed for the most frequently used 96-well and 6-well plates by physical measurement.The human breast cancer cell line,MDA-MB-231,was used to verify the differences in the three cell irradiation methods on cell radiosensitivity by usingcellproliferationandclonogenicassays.Theaimofthe present study was to establish and validate an accurate and convenient cell irradiation method,thereby providing an experimental basis and theoretical support for the study of radiosensitivity of tumor cells.

在黔西南州海拔1 700米以上的山区茶园，通常只有1个虫口高峰期，且峰期较长。一般4月下旬开始虫口数逐渐上升，6～10月虫口量最高，危害整个夏、秋茶，茶树受害严重。

2 Materials and methods

2.1 Radiotherapy equipment

The radiotherapy equipment included a linear accelerator(Synergy VMAT,Elekta),a dosimeter(UNIDOS E),a plane-parallel ionization chamber(34001,PTW,Germany),and a RW3 slab phantom(T29672,PTW,Germany,that is solid water)with thicknesses of 0.1,0.2,0.5,and 1 cm and dimensions of 30×30 cm2.

2.2 Simulated ‘cell culture plate’system

3.2.1 Comparison of proliferation of cells

2.3 Irradiation methods

The 6 MV X-ray was used,the depth of the maximum dose is 1.5 cm below the water,and D20/10=0.585.The thickness of the irradiated material required is 1.5 cm for the 6 MV X-ray beam.The simulated ‘cell culture plate’’system was irradiated at five different preset doses:2,4,6,8,and 10 Gy,by the three methods.Each measurement was repeated five times.

(i) Method G0B0F40 The 6 cm solid water(including the thickness of ware bottom and the gap between the bed and the bottom of the cell culture plate)was placed below the plane-parallel ionization chamber.This solid water is used as a back-scattering material.There were no additional build-up materials,as shown in Fig.2a.

水平三角形的第（4）浪之后，进入一气呵成的第（5）浪，至2015年6月7559点结束。当初将2013年6月底的低点划分作第（4）浪终点，将（4）浪D看作（5）浪1——这是常见的错误——因此才有后来的（5）浪5预期。7559点结束第（5）浪，这是循环浪III的顶点，因此后面的调整属于循环浪IV。其中，第一个浪A可能会于2019年结束，特别是当国证A指跌至上升通道的下轨时，配合波浪理论，将成为循环浪IV第一个买点。

Fig.1 A cross section of the simulated ‘cell culture plate’system.A Ware bottom;B culture medium;C the phantom of building cavity;D cavity;E ware cover;F the gap between the bed and the bottom of the cell culture plate.(A,B,and E are all built of solid water).In the real cell irradiation experiment,F is escapable and filled by using solid water.Thus,the gap is filled by using solid water in the simulation.C is built of polystyrene,in which the density is nearly equal to that of air,in order to build the cavity.G is used to put the plane-parallel ionization chamber

Table 1 Speci fic parameters of cell culture plastic ware

Ware height(cm) Culture medium height(cm) Cavity height(cm)96-well plate 1.1 0.6 0.5 6-well plate 1.7 0.3 1.4

Fig.2 Three irradiation methods for the simulated ‘cell culture plate’system.a G0B0F40(a certain thickness of solid water(H)is placed on the below simulated ‘cell culture plate’system,to constructa6 cm thicknessoftheback-scattering materials).b G0B1.5F40(a certain thickness of solid water(I)is placed on the above simulated ‘cell culture plate’system,to construct a 1.5 cm thickness of the back-scattering materials;a certain thickness of solid water(H)is placed on the below simulated ‘cell culture plate’’system,to construct a 6 cm thickness of back-scattering materials).c G180B1.5F40(a certain thickness of solid water(I)is placed on the below simulated ‘cell culture plate’system,to construct a 1.5 cm thickness of the back-scattering materials).Note:H stands for the additional back-scattering materials;I stands for the additional adding build-up materials;For G,B,F with the name of Method G0B0F40,G0B1.5F40,G180B1.5F40,G stands for gantry degree;B stands for the thickness of build-up materials;and F stands for field size

(iii) Method G180B1.5F40 Solid water of 1.5 cm thickness(including the thickness of ware bottom and the gap between the bed and the bottom of the cell culture plate)was placed below the plane-parallel ionization chamber.A certain thickness of solid water was placed below the simulated ‘cell culture plate’system,in order to construct a 1.5 cm thickness of the build-up materials,as shown in Fig.2c.

2.4 Cell culture

The breast cancer cell line,MDA-MB-231,was purchased from the Cell Bank of the Chinese Academy of Sciences(Shanghai,China)and stored in the Central Laboratory of Shanghai Tenth People’s Hospital.The MDA-MB-231 cells were cultured in Dulbecco’s modi fied Eagle’s medium(DMEM)supplemented with 10%fetal bovine serum(FBS)at 37°C in an incubator with 5%CO2 and 95%humidity.

(ii) Method G0B1.5F40 The 6 cm solid water(including the thickness of ware bottom and the gap between the bed and the bottom of the cell culture plate)was placed below the plane-parallel ionization chamber.This solid water is used as a back-scattering material.Solid water of 1.5 cm thickness was then placed over the plane-parallel ionization chamber(including the thickness of ware cover and the height of culture medium).A certain thickness of solid water was placed on the above simulated ‘cell culture plate’’system,in order to construct a 1.5 cm thickness of build-up materials,as shown in Fig.2b.

2.5 Cell veri fication test

2.5.1 Cell proliferation assay

Cell proliferation was assessed by using the Cell Counting Kit(CCK)-8 assay(Shanghai Yisheng Biotechnology Co.,Ltd,Qingdao,China)and a microplate reader(BioTek Company,VT,USA).Brie fly,the hybrids andtheir parental counterparts were plated at 1200 cells/well in 96-well plates.After incubation for the indicated time,CCK-8 was added at 200 μl/well,and irradiation with 6 MV X-ray of 4 Gy was performed,with a source-axis distance(SAD)of 100 cm by using three different physical methods.Given the length of the paper,only the data for 4 Gy were provided and discussed;the other data will be discussed more extensively in another paper.The optical density was measured at 450 nm by using a microplate reader(BioTek Company,VT,USA).

Table 2 Actual measured doses of two different cell culture plastic wares for Method G0B0F40,G0B1.5F40 and G180B1.5F40

All the measure dose are presented as mean±SD.Deviation is presented as

Method Preset dose(Gy)2 4 6 8 10(a)96-well plate G0B0F40 Measure dose(Gy) 1.9090±0.0010 3.8092±0.0008 5.7188±0.0008 7.6274±0.0023 9.5078±0.0114 Difference(%) -4.55 -4.77 -4.69 -4.66 -4.92 G0B1.5F40 Measure dose(Gy) 1.9990±0.0007 3.9890±0.0007 5.9940±0.0016 8.0012±0.0013 9.9960±0.0019 Difference(%) -0.05 -0.28 -0.10 0.02 -0.04 G180B1.5F40 Measure dose(Gy) 1.9628±0.0004 3.9132±0.0024 5.8848±0.0023 7.8556±0.0047 9.8202±0.0034 Difference(%) -1.86 -2.17 -1.92 -1.81 -1.80(b)6-well plate G0B0F40 Measure dose(Gy) 1.8572±0.0011 3.7076±0.0015 5.5754±0.0029 7.4468±0.0018 9.3062±0.0036 Difference(%) -7.14 -7.31 -7.08 -6.92 -6.94 G0B1.5F40 Measure dose(Gy) 2.0062±0.0004 4.0000±0.0016 6.0020±0.0012 8.0058±0.0033 9.9876±0.0086 Difference(%) 0.31 0.00 0.03 0.07 -0.12 G180B1.5F40 Measure dose(Gy) 1.9316±0.0021 3.8694±0.0024 5.8184±0.0039 7.7708±0.0047 9.7138±0.0052 Difference(%) -3.42 -3.27 -3.03 -2.87 -2.86

2.5.2 Clonogenic assay

现今中国的高校教学较之过去发生了极大的变化，高校教师教学任务繁重，教学对象复杂，教学要求提高。而与之形成鲜明反差的是，高校教师教学投入相对减少，高校教学面临着前所未有的挑战。当前的高校教学呈现以下特点。

3 Results

3.1 Simulated ‘cell culture plate’system irradiation

Fig.3(Color online)a the cell growth curves for the irradiated MDA-MB-231 cells with Method G0B0F40,Method G0B1.5F40,and Method G180B1.5F40(n=6 per group,values represent the mean±SD);b the comparison of the surviving fraction of the MDA-MB-231 cells after the three methods.All data are presented as the mean±SD from four replicate wells

The actual measured doses in the ‘cell culture plate’’system(96-well and 6-well plates)using the methods G0B0F40,G0B1.5F40,and G180B1.5F40 are shown in Table 2.For the 96-well plate,at each preset dose the range of the differences between the measured dose and preset dose was between-0.28 and 0.02% with Method G0B1.5F40,between-2.17 and-1.80%with Method G180B1.5F40,and the maximum difference was between-4.92 and-4.55%with Method G0B0F40.For the 6-well plates,at each preset dose,the range of the differences between the measured dose and the preset dose was between 0.31 and-0.12%with Method G0B1.5F40,between-3.42 and-2.86%with Method G180B1.5F40,and the maximum difference was between-7.31 and-6.92%with Method G0B0F40.In addition,the dose differences in the 96-well plates were generally lower than that of the 6-well plate for Method G0B0F40 and Method G180B1.5F40.Overall,for each preset dose,the actual measured dose was closest to the preset dose with Method G0B1.5F40 and lower than the preset dose with Method G180B1.5F40.Under the conditions of Method G0B0F40,the measured actual dose was lowest,with the maximum deviation from the preset dose.Therefore,the above results show that Method G0B1.5F40 was chosen as the optimal method for cell irradiation.

3.2 Comparison of cell veri fication test

The cell culture plate system was made up of ware cover,cavity,culture medium,and ware bottom.As the density of the solid water,which is often adopted as the clinical homogeneousphantom thatisclosetothatofthe cellculture plate,the system was built up and simulated by the solid water.Thecavitywasbuiltoffourpiecesofpolystyrenewith a density merely equal to that of air that was placed on the solid water.The well wall was ignored,as shown in Fig.1.Thespeci ficparametersofthecellcultureplatesimulatedby solid water are shown in Table 1.

MDA-MB-231 cells were irradiated with 4 Gy,and the cell growth curves were measured by CCK-8 assay after irradiation using Method G0B0F40,G0B1.5F40,and G180B1.5F40,as shown in Fig.3a.There was no signi ficant difference in the survival rate of MDA-MB-231 cells at 24 h after attachment between the three methods.The cells entered the logarithmic growth phase at 48 h after attachment.The difference appeared at 72 h after attachment,and it became larger as time progressed.The OD value with Method G0B0F40 was highest,followed by Method G180B1.5F40,and thelowestforMethod G0B1.5F40,which indicated that the actual irradiated dose of cells was lower than that with the other two methods.Method G0B1.5F40 had the lowest OD value and the lowest cell survival rate;the actual irradiated dose of cells in this method was higher than that of Method G0B0F40 and Method G180B1.5F40.It was also confirmed that the actual irradiated dose of cells for Method G0B1.5F40 was the highest overall,followed by Method G180B1.5F40 and then Method G0B0F40,in which the actual irradiated dose of cells was the lowest.

MDA-MB-231cellsinthelogarithmicgrowthphasewere harvested and 500 cells/well were reseeded in 6-well cell culture plates.After incubation for 24 h,the cells were irradiated with 6 MV X-ray of 4 Gy,a source-axis distance(SAD)of 100 cm,by using the three different physical methods.Similarly,onlythedatafor4 Gywereprovidedand discussed in this experiment.After 9 days,medium-free cells were fixed with absolute ethyl alcohol for 10 min,stainedwith0.5%crystalvioletfor15 min,andthenwashed three times with PBS.The colonies containing＞50 cells were counted.The plating efficiency(PE)and surviving fraction(SF)were calculated from the following formulae:

3.2.2 Comparison of the clonogenic capacities of cells

The dose differences of the 96-well plates were generally less than that of the 6-well plate for methods G0B0F40 and G180B1.5F40.For Method G0B0F40,the thickness of the build-up materials was 0.6 cm in the 96-well plate and 0.3 cm in the 6-well plate.Thus,the measured dose was closer to the preset dose:The dose difference was smaller for the 96-well plate,but not for the 6-well plate.For Method G180B1.5F40,the thickness of back-scattering material was 0.6 cm in the 96-well plate and 0.3 cm in the 6-well plate.In a 5 cm thickness of back-scattering material range[13],the thickness of the back-scattering material is thicker,there are more back-scattering photons,and the back-scattering effect of the dose was larger.Thus,the measured dose was closer to the preset dose,and the dose difference was smaller for the 96-well plate,but not for the 6-well plate.This also demonstrated that it was important to add the appropriate thickness of the build-up and backscattering materials for cell irradiation according to the energy of the linear accelerator.

4 Discussion

As the cells are protected from contamination,the culture medium cannot be filled generally;a certain gap should be left between the upper surface of the culture medium and the culture plastic ware cover,and it is inescapable in the process of cell culture.This gap will cause the cavity effect of X-ray irradiation,which has been reported in several studies[15,16].A study by Xiao et al.[17].has suggested that the cavity effect was lower when the air cavity was located in the deeper region within the tissues than when it is located in the shallower region within tissues.For the methods G0B1.5F40 and G180B1.5F40,there is a 1.5 cm thickness of build-up materials,so the cavity effect was lower than that of G0B0F40.A study by Wang et al.[18].suggested that when the area of the radiation field was greater than that of sagittal interface of the nasopharynx cavity,the nasopharyngeal cavity has very little in fluence on the dose distribution of the coaxial penetrating field,and it can therefore be neglected.In this study,a 40×40 cm2radiation field was adopted for the three methods[19]and the area of the radiation field was greater than that of cavity,so the cavity effect can be ignored and the actual measured dose would be closer to the preset dose;that is,the cavity effect can be ignored for method G0B1.5F40.Thus,Method G0B1.5F40 represents the optimal method for cell irradiation.

As can be seen from Fig.3b,obvious differences in the surviving fraction were found for cells after the three differentirradiation methods.Formethods G0B0F40,G180B1.5F40,and G0B1.5F40,SF(%)was 66.31±7.10,31.52±5.27,and 25.12±5.21,respectively.In addition,the surviving fraction of the cells for Method G0B1.5F40 was signi ficantly lower than that of Method G0B0F40 and slightly lower than that of Method G180B1.5F40.This showed that the actual irradiated dose of cells was highest with Method G0B1.5F40 and closest to the preset dose.When using this method,the radiosensitivity applied to the cell was accurate.

It is not clear if the actual irradiated dose for the cells reaches the preset dose,as the results from different irradiation methods are contradictory.Although many research articles have reported data on cellular radiosensitivity,the published descriptions of the irradiation methods tend to be imprecise[12,13].In this study,the results showed that no matter what type of cell culture plate was used,the best irradiation method,in which the irradiated dose was closest to the preset dose,occurred after our Method G0B1.5F40 above.For method G0B1.5F40,there was a 1.5 cm thickness of build-up materials(including the thickness of the ware cover and the height of culture medium)on the upside of the cell,the irradiated dose of cells is at the point of maximum dose,and the 6 cm thickness of the back-scattering materials(including the thickness of ware bottom and the gap between the bed and the bottom of the cell culture plate)at the back of the cell increases back-scattering.In this case,a millimeter of difference in the height of the culture medium has little impact on the experiment.For Method G0B0F40,although the 6 cm thickness of solid water(including the thickness of the ware bottom and the gap between the bed and the bottom of the cell culture plate)can increase back-scattering,there is insuf ficient build-up of materials(for 6 MV,the thickness of the buildup materials is 1.5 cm)on the upside.In this case,the actual irradiated dose of cells falls within the range of the high energy X-ray dose build-up region,and the actual irradiated dose is low.The millimeter of difference in the height of the culture medium in the actual experiment leads to a larger difference in the actual irradiated dose of cells.In Method G180B1.5F40,although there is a 1.5 cm thickness of build-up materials(including the thickness of ware bottom and the gap between the bed and the bottom of the cell culture plate),there was no material in the emission direction of the X-ray to increasing back-scattering,which results in a lower actual irradiated dose of cells than the preset dose.This occurs because the actual cell irradiation is not feasible.Therefore,it is important to add a 1.5 cm thickness of build-up materials(including the thickness of ware cover and the height of the culture medium)into the area above the cells after irradiation of the cells with 6 MV X-ray.In addition,a certain thickness of back-scattering material should be added to the area under the tissue culture plastic ware.In a study by Zheng et al.[14],who used a 6 MV X-ray linear accelerator,it was suggested that at least 5 cm thickness of solid water should be added as the back-scattering material.Therefore,in this paper,a 6 cm thickness of solid water was used as the back-scattering material.The thickness of back-scattering material in fluences the cell dose,but this is not discussed in this paper.

2) 储罐泡沫喷射口设置在罐壁顶部，在2个泡沫喷射口的中间区域存在一定范围的喷射盲区，泡沫不能全部覆盖火灾区域，内浮顶储罐固定灭火系统覆盖范围盲区如图1所示，其中V-1是泡沫喷射的中间盲区，V-2是泡沫喷射边缘盲区。发生火灾区域的热气流上升，冷空气从浮船上部位置下降，泡沫散射降落过程中部分泡沫液被卷吸、漂散至罐外；由于受到火焰的高温作用，大量泡沫液到达油面之前已破裂、分解甚至气化蒸发，泡沫液损失严重，很难喷射到火源根部，达不到隔离空气的目的。调研日本的储罐灭火试验结果，泡沫灭火系统中损耗的泡沫液量占61%，蒸发的泡沫液量占9%，落在油品液面发挥灭火功能的泡沫仅占30%[8]。

由图6 b)可知，通过数值计算模拟、Peck公式计算与实地监测得到的地表沉降曲线变化趋势基本一致，均呈 W形分布。其中左线隧洞上方的地表沉降值略大于右线位置，因为左线开挖时，土体应力再次发生变化，地基进一步发生沉降，且由于右线通过壁后注浆等支护措施，已基本弥补地层损失，故右线位置二次沉降值略小。同时随着盾构机向前推进，各测点的沉降值均逐渐增大。

In summary,when the cell will be irradiated,we must pay attention to the in fluence of the build-up effect and cavity effect in the future.

虚宁寺的“静”不仅仅是对于进寺院游览的信众和游客而言，对于住寺僧团及工作人员来说，亦如此。这主要是源于虚宁寺特别的管理形式。

According to the experience of proliferation and the colony formation assays for MDA-MB-231 cells from literature reports[20–22],the cell proliferation assay and clonogenic assay of MDA-MB-231 cells were performed to verify the difference of the three methods in this paper.For the cell proliferation assay,we found no signi ficant difference in the survival rate of cells at 24 h after cell attachment for the three irradiation methods.This mainly resulted from the fact that cell apoptosis was not clear at this point and the obvious effect of radiotherapy on cell viability was small.After 48 h,the radiotherapy started to exert an effect on the cells.The dose was higher,and the cell survival rate was lower at 72 h after cell attachment.The cell survival rate was highest in Method G0B0F40,because the actual irradiated dose of cells was lowest,followed by Method G180B1.5F40,and then Method G0B1.5F40.Compared with the other two methods,the actual measured dose in Method G0B1.5F40 was closest to the preset dose and the dose was highest,which resulted in the lowest cell survival rate.The cell growth curves demonstrated that there were signi ficant differences for the three cell irradiation methods after 72 h.For the cell clonogenic assay,we can see that SF was highest for Method G0B0F40, second highest for Method G180B1.5F40,and lowest for Method G0B1.5F40 after 9 days.This is the dose of Method G0B1.5F40 that was higher than that of other methods,and its dose was closest to the preset dose.This confirmed that method G0B1.5F40 was the optimal method for cell irradiation.From the above two experiments,which showed no signi ficant dose difference in the cell experiments after 48 h for the three methods,but the dose difference was obvious in cell experiments(e.g.,the clonogenic assay)after 48 h for the three methods.Each cell experiment lasts for a different time;for example,the cell clonogenic assay usually takes longer than 9 days.Thus,method G0B1.5F40 should be chosen at the time of cell experiment.Therefore,an accurate cell irradiation method must be established before conducting a cell radiosensitivity test.An irradiation dose that is too high or too low will affect the experimental results,which will be detrimental to the comparison of the relevant experimental results of authors from different research laboratories.

In this study,three different irradiation methods were used to measure the physical doses of the cell for the simulated ‘cell culture plate’system.It was demonstrated through the growth curve and colony-forming efficiency of MDA-MB-231 cells that Method G0B1.5F40 was the best cell irradiation method.In the cell culture process,the SCD,build-up and back-scattering effect,cavity effect,and the feasibility and convenience of the operation should be fully considered with the linear accelerator.The most accurate and effective cell irradiation method should always be used,which will reduce the dose differences and instability factors and provide a greater accuracy and comparability for laboratories conducting research into cellular radiosensitivity.

又如《荆轲刺秦王》的开头写道“秦将王翦破赵，虏赵王，尽收其地，进兵北略地，至燕南界”，交代了故事发生的背景，把太子丹和荆轲这两个主要人物放在秦、燕冲突的大背景下来表现，面对危急的形势，“太子丹恐惧”、荆轲“愿得谒之”，二人的性格差异由此可见一斑。“太子迟之。疑其有改悔，乃复请……荆轲怒，叱太子曰……”这一矛盾冲突，用太子丹的浮躁多疑、谋事不周鲜明地反衬出荆轲的沉稳机智等性格特征。“荆轲廷刺秦王”这一部分是全文的高潮部分，是矛盾冲突最尖锐的时候，荆轲和秦王之间展开了一场你死我活的搏斗，这一惊心动魄的斗争场面展现了荆轲大无畏的英雄气概。

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