A549细胞超微结构体视学研究
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摘要
研究背景和目的
A549细胞来源于人肺泡细胞癌,由Giard D J等于1973年建立。该细胞既具有肺癌恶性肿瘤细胞的特性,同时又具有肺泡Ⅱ型上皮细胞的特性和表型。故在研究中A549细胞被广泛用于肺癌的发生、发展、诊断及治疗研究,也被用于正常人肺泡Ⅱ型上皮细胞的发生、分化及其结构和功能的研究。根据Pubmed查询,到目前为止,使用A549细胞进行研究的英文报道达6200余篇；CNKI收录的利用A549细胞进行研究的中文文献多达4600余篇,且中英文献数均逐年呈倍数级上升。然而,这些报道主要集中于A549细胞功能方面的研究。
众所周知,细胞形态变化是其功能变化的基础,而细胞功能方面的变化又将影响细胞形态结构。要想提高对细胞功能的理解和认识必须深化对细胞形态和结构的认识。以往对形态的认识和研究多局限于镜下的定性观察。然而,对A549细胞的形态结构研究,无论是光学显微镜之“镜下所见”,还是扫描电镜抑或透射电镜下的超微结构观察,采用的均为经典的视觉描述来反映细胞的形态结构及其变化,且都局限于二维水平,缺乏形态结构的量化概念,缺乏三维结构水平的定量研究。而且由于各种视觉错觉的作用,如佐勒错觉和埃宾豪斯错觉等,有时甚至会得出完全错误的结论。从结构形态来看二维图像也只是显示三维结构的各种剖面,例如对于二维切片中的圆形结构并不一定意味着其三维结构为球体,它可能是球体,也可能是椭圆、圆管状或不规则结构等。从结构数量来看,二维图像一般只能显示三维结构的一部分,有些结构数目少,分布不均匀时,在二维图像中往往看不到。因此,二维图像上所见的细胞形态,诸如各种结构的形状、大小、多少都发生了变化,单纯描述镜下所见并不全面,也不完善。
体视学是由二维结构信息定量推论三维结构信息的介于形态学与数学之间的新兴学科。应用体视学原理最终可测得细胞的三维结构参数,使人们能从三维水平定量地阐明细胞的结构。例如,细胞中线粒体的体密度能反映出线粒体相对体积大小,线粒体数密度反映了线粒体数量多少,线粒体内膜表面积密度可反映线粒体内膜表面积大小,线粒体的平均自由程则反映出线粒体与线粒体之间的空间平均距离,或者反映了线粒体是密集还是稀少,而线粒体的曲度、球度、轴比等则从不同方面反映了线粒体的形态等。其他诸如细胞、核、各种细胞器及细胞中的包含物等的三维结构特征都可用相应的体视学参数来定量描述。
本研究利用光镜与电镜拍摄细胞的图像,采用图像分析软件Image-Pro Plus6.0对A549细胞的图像进行分析,并应用体视学的原理和方法,对A549细胞及其细胞核内的核仁、常染色质、异染色质以及细胞质内的线粒体、溶酶体、板层小体及囊泡等超微结构的三维形态特征进行量化,从三维水平定量揭示A549细胞超微结构的特点。在研究过程中,我们还对影响参数结果的因素进行了校正如细胞收缩的校正及测试系统的标定等。
方法
A549细胞株和HBE细胞株由病理教研室液氮保存。
1.A549细胞光镜及电镜制样收缩系数测试
(1)分别用等渗重悬、涂片HE染色、涂片巴氏染色、石蜡包埋3μm切片HE染色及透射电镜制样半薄(3μm)切片HE染色5种常规方法处理细胞；
(2)用Image-Pro Plus 6.0图像分析软件测试每组细胞的平均直径,然后以A549培养之等渗重悬细胞之平均直径为基准计算细胞的收缩系数。
2.电镜图像“bar”尺长度的误差分析
(1)用光栅复型标定测试软件并用软件按标定后的标准尺度测量3500倍、8000倍和15000倍电镜图像上对应给出的10μm、5μm和2μm“bar”尺的真实长度,用测得的“bar”尺真实长度与图像所标的长度进行比较分析；
(2)计算电镜图像“bar”尺所给的长度与其真实长度间的绝对误差和相对误差,比较不同放大倍数图像上“bar”尺所给的长度与其真实长度间的相对误差差异程度。3.A549细胞超微结构体视学研究
(1)透射电镜下观察A549细胞内细胞器的结构并予辨认；
(2)用Image-Pro Plus 6.0图像分析软件设计测试系统,应用体视学原理和方法,测试A549细胞超微结构的体视学参数包括：细胞核体密度(VVn)、细胞浆体密度(VVcyt)、细胞核表面积密度(SVn)、核内常染色质体密度(VVeuc)、异染色质体密度(VVhet)、核仁体密度(VVnu)、核仁表面积密度(SVnu)、线粒体体密度(VVmi)、线粒体表面积密度(SVmi)、线粒体数密度(NVmi)；溶酶体体密度(VVlys)、溶酶体表面积密度(SVlys)、溶酶体数密度；板层小体体密度(Vlam)、板层小体表面积密度(SVlam)、板层小体数密度(VVlam)；囊泡体密度(VVves)、囊泡表面积密度(SVves)、囊泡数密度(NVves)；细胞浆平均体积(vcyt)、细胞核平均体积(vn)及平均表面积(sn)；核仁平均体积(vn)及平均表面积(snu)；线粒体的平均体积(vmi)、平均表面积(smi)及数量(nmi);溶酶体的平均体积(vlys)、平均表面积(slys)及数量(nlys)；板层小体的平均体积(nlam)、平均表面积(slam)及数量(nlam)；囊泡的平均体积(vves)、平均表面积(sves)及数量(nves)。上述参数中的细胞浆平均体积(vcyt)、细胞核平均体积(vn)及平均表面积·(sn),核仁平均体积(vn)及平均表面积(snu),线粒体的平均体积(vmi)、平均表面积(smi)及数量(nmi),溶酶体的平均体积(vlys)、平均表面积(slys)及数量(nlys),板层小体的平均体积(nlam)、平均表面积(slam)及数量(nlam)及囊泡的平均体积(vves)、平均表面积(sves)及数量(nves)是指一个A549细胞内对应细胞器参数的绝对含量。细胞核及浆的密度参数以细胞为参照空间,核仁、常染色质及异染色质的密度参数以细胞核为参照空间,线粒体、溶酶体、板层小体及囊泡结构的密度参数以细胞浆为参照空间。
4.A549细胞和HBE细胞形态结构的差异分析
(1)光学显微镜、倒置显微镜、扫描电镜及透射电镜下观察A549细胞和HBE细胞的形态特点；
(2)通过Image-Pro Plus 6.0图像分析软件鼠标勾描每幅图像中的细胞、细胞核和核仁的轮廓,分别测试细胞、细胞核及核仁的面积和周长,并计数细胞内的胞核数及核内的核仁数,以细胞为参照空间计算HBE细胞和A549细胞核及核仁形态的体视学参数：包括细胞核体密度(VVn)、细胞核表面积密度(SVn)、细胞核表面积与体积比(RSVn)、细胞核数密度(NVn)、细胞核平均体积(vn)、细胞核平均表面积(Sn)和核浆比(Rnp)；细胞核仁的体视学参数包括核仁体密度(VVnu)、核仁表面积密度(SVnu)、核仁表面积与体积比(RSVnu)、核仁数密度(NVnu)、核仁平均体积(Vnu)、核仁平均表面积(Snu)共13种体视学参数的数值。
(3)以HBE细胞核及核仁形态参数值为基准,计算A549细胞核及核仁形态的正负偏离度。
结果
1.A549细胞光镜及电镜制样的收缩系数测试
(1)A549细胞平均直径的测试结果
等渗液重悬A549细胞的平均直径为14.9302μm,范围在10.0248-19.3552μm之间,变异系数为0.0998；涂片HE染色A549细胞的平均直径为9.8775μm,范围在7.5651-14.4611μm之间,变异系数为0.1009；涂片巴氏染色A549细胞的平均直径为9.9772μm,范围在7.6697-14.5422μm之间,变异系数为0.1071；石蜡包埋3μm厚切片HE染色A549细胞的平均直径为10.2914μm,范围在6.0952-16.1046μm之间,变异系数为0.1419；透射电镜半薄切片HE染色A549细胞的平均直径为10.5857μm,范围在3.6853-20.3462μm之间,变异系数为0.2653。
(2)A549细胞收缩系数的计算结果
以等渗重悬细胞的平均直径为基准,涂片HE染色、涂片巴氏染色、石蜡包埋3μm厚切片和透射电镜制样环氧树脂包埋3μm厚切片HE染色后细胞的收缩系数分别为：0.66、0.67、0.69和0.71。经过涂片HE染色、涂片巴氏染色、石蜡包埋切片HE染色和电镜制样半薄切片HE染色后A549细胞均有较明显的收缩,其平均直径与等渗重悬的A549细胞的平均直径比较差异均有显著性(P=0.000)。
2.电镜图像“bar”尺长度误差的测试
(1)电镜图像上“bar”尺的真实长度测试结果
3500倍图像长度为10μm“bar”尺的真实长度均值为10.3137μm,范围在10.3065-10.3410μm之间,变异系数为0.0011；8000倍图像长度为5μm“bar”尺的真实长度均值为5.1424μm,范围在5.1360-5.2079μm之间,变异系数为0.0024；15000倍长度为2μm“bar”尺的真实长度均值为1.9265μm,范围在1.9132-1.9337μm之间,变异系数为0.0046。3500倍、8000倍和15000倍电镜图像上“bar”尺的长度与其真实长度间的比较差异均有显著性(P=0.000)。
(2)电镜图像上“bar”尺长度与其真实长度间的绝对误差计算结果
3500倍图像“bar”尺长度与其真实长度间的绝对误差均值为0.3137μm,范围在0.3065-0.3410μm之间,变异系数为0.0363；8000倍图像“bar”尺长度与其真实长度间的绝对误差均值为0.1424μm,范围在0.1360-0.2079μm之间,变异系数为0.0880；15000倍图像“bar”尺长度与其真实长度间的绝对误差均值为0.0735μm,范围在0.0663-0.0868 u m之间,变异系数为0.1210。
(3)电镜图像上“bar”尺长度与其真实长度间的相对误差计算结果
3500倍图像“bar”尺长度与其真实长度间的相对误差均值为3.04%,范围在2.97%-3.30%之间,变异系数为3.62%；8000倍图像“bar”尺长度与其真实长度间的相对误差均值为2.77%,范围在2.65%-3.99%之间,变异系数为8.30%；15000倍图像“bar”尺长度与其真实长度间的相对误差均值为3.82%,范围在3.43%-4.54%之间,变异系数为12.56%。3500倍、8000倍和15000倍电镜图像“bar”尺长度与其真实长度间的相对误差三种不同放大倍数整体比较差异有显著性(F=58.862,P=0.000)。3500倍、8000倍和15000倍电镜图像上“bar”尺长度与其真实长度间的相对误差两两比较差异均有显著性(P=0.000)。
3.A549细胞三维超微结构的形态分析
(1)A549细胞透射电镜下超微结构观察
A549细胞表面有大量的短小微绒毛。细胞质丰富,内有各种细胞器如线粒体、溶酶体、内质网、高尔基复合体、板层小体、粘液空泡和微囊等。线粒体在细胞中分布不均一,呈极性分布；溶酶体大部分属于自噬溶酶体,由单位膜包绕,其内含有残余的细胞器；板层小体为圆形或卵圆形,内部有呈平行或螺纹状排列的板状结构；微囊的腔面见微绒毛,腔内有不同电子密度的分泌物；粘液空泡往往几个或一群密集分布,其电子密度较低。局部区域内质网、高尔基复合体结构丰富。细胞核大,核形不规则,可见极不规则核；核膜皱褶多；染色质细密,常染色质占大部分；核内可见一个或多个核仁。观察、辨认细胞电镜图像发现板层小体和微囊两种超微结构,可作为鉴定A549细胞的佐证之一
(2)A549细胞超微结构形态的量化特点
①A549细胞核及浆形态的量化特点
VVn均值为0.2789,其95%的可信区间在0.2451-0.3129之间,变异系数为0.2191；VVcyt均值为0.7211,其95%的可信区间在0.6871-0.7549之间,变异系数为0.0847；Rnp为0.63；SVn均值为0.1934μm-1,其95%的可信区间在0.1785-0.2075μm-1之间,变异系数为0.2004；RSVn为0.67；vn均值为465.76μm3,其95%的可信区间在409.25-522.27μm3之间,变异系数为0.2091；vcyt均值为1204.2μm3,其95%的可信区间在1147.7-1260.7μm3之间,变异系数为0.0847；sn均值为322.92μm2,其95%的可信区间在298.76-347.08μm2之间,变异系数为0.2004。
②A549细胞核内核仁、常染色质及异染色质形态的量化特点
VVnu。均值为0.0761,其95%的可信区间在0.0739-0.0781之间,变异系数为0.0571；SVnu均值为0.1508μm-1,其95%的可信区间在0.1424-0.1596μm-1之间,变异系数为0.1518,因此A549细胞核仁的表面积与体积比为1.98。vnu均值为35.623μm3,其95%的可信区间在34.497-36.749μm3之间,变异系数为0.0571；snu均值为70.517μm2,其95%的可信区间在66.521-74.513μm2之间,变异系数为0.1518。VVeuc均值为0.7225,其95%的可信区间在0.7097-0.7363之间,变异系数为0.0372；Veuc均值为337.85μm3,其95%的可信区间在331.73-343.97μm3之间,变异系数为0.0372。VVher均值为0.2012,其95%的可信区间在0.1883-0.2137之间,变异系数为0.1164；Vhet均值为94.114μm3,其95%的可信区间在88.047-100.18μm-3之间,变异系数为0.1164。
③A549细胞线粒体形态的量化特点
VVmi均值为0.0460,其95%的可信区间在0.0443-0.0477之间,变异系数为0.0883；SVmi,均值为0.5988μm3,其95%的可信区间在0.5045-0.6935μm-1之间,变异系数为0.5638；NVmi均值为0.3003μm-3,其95%的可信区间在0.2296-0.3704μm-3之间,变异系数为0.4977。vmi均值为61.910μm3,其95%的可信区间在58.882-64.938μm3之间,变异系数为0.0883；smi均值为1001.67μm2,其95%的可信区间在888.09-1115.3μm2之间,变异系数为0.5683；一个A549细胞含nmi约501个,其95%的可信区间在384-618个之间,变异系数为0.4977。
④A549细胞溶酶体、板层小体和囊泡形态的量化特点
VVlan均值为0.0250,其95%的可信区间在0.0162-0.0338之间,变异系数为0.6242；VVlys均值为0.0129,其95%的可信区间在0.0091-0.0119之间,变异系数为0.5177；VVves均值为0.0390,其95%的可信区间在0.0397-0.0401之间,变异系数为0.0466。SVlam均值为0.3565μm-1,其95%的可信区间在0.2715-0.4425μm-1之间,变异系数为0.6432；SVlys均值为0.1763μm-1,其95%的可信区间在0.0987-0.2533μm-1之间,变异系数为1.1744：SVves均值为1.1612μm-1,其95%的可信区间在1.0485-1.2735μm-1之间,变异系数为0.2591。NVlam均值为0.5691μm-3,其95%的可信区间在0.4337-0.7043μm-3。之间,变异系数为0.7127；NVlys均值为0.1289μm-3,其95%的可信区间为0.0874-0.1706μm-3之间,变异系数为0.4624；NVves均值为10.424μm-3,其95%的可信区间在8.7206-12.274μm-3之间,变异系数为0.3178；vlam值为76.820μm3,其95%的可信区间在62.384-91.257μm-3之间,变异系数为0.6242；vlys均值为21.694μm-3,其95%的可信区间在15.474-27.914μm-3之间,变异系数为0.5177；vves均值为65.241μm3,其95%的可信区间在63.774-66.708μm3之间,变异系数为0.0406；slam均值为428.68μm2,其95%的可信区间在325.73-531.63μm2之间,变异系数为0.6432；slys均值为212.04μm2,其95%的可信区间在119.06-305.02μm2之间,变异系数为1.1744；sves均值为1396.2μm2,其95%的可信区间在1261.1-1531.3μm2之间,变异系数为0.2591；一个A549细胞内,nlam约含950个,其95%的可信区间在724-1175个之间,变异系数为0.7127.nlys约含215个,其95%的可信区在146-284个之间,变异系数为0.4624；nves约含17409个,其95%的可信区间在14564-20253个之间,变异系数为0.3178.
4.A549和HBE细胞形态结构差异分析
(1)倒置显微镜和细胞爬片HE染色观察结果
A549细胞和HBE细胞均为大小不一、形态多样。A549以梭形、多角形细胞为主,散在、单层排列,HBE细胞以多边形上皮样细胞为主,梭形细胞只占极少数,镶嵌排列。HBE细胞形态相对A549较一致。
(2)扫描电镜及透射电镜下A549细胞和HBE细胞形态观察结果
扫描电镜下：HBE细胞和A549细胞的表面可见细胞皱褶、突起和细小均匀分布的纤毛。透射电镜观察,HBE细胞和A549细胞中可见不规则细胞核,其内见一个或多个核仁。
(3)A549细胞及HBE细胞核及核仁形态差异定量分析结果
以HBE细胞核及核仁的形态参数为基准,A549细胞核体密度、核表面积密度、核数密度和核表面积与体积比的负偏离度分别为21.14%、27.96%、50%和32.87%,A549细胞核平均体积、核平均表面积和核浆比的正偏离度分别为146.98%、59.65%和12.90%,A549细胞核仁体积密度、核仁表面积密度、核仁数密度及核仁表面积与体积比的负偏离度为7.81%、33.63%、43.68%和36.64%,A549细胞核仁的平均体积、核仁平均表面积的正偏离度分别为53.49%和13.48%。
结论
1.A549细胞经过涂片HE染色、涂片巴氏染色和石蜡包埋3μm切片HE染色及透射电镜制样半薄(3μm)切片HE染色后会明显收缩,收缩系数分别为：66%、67%、69%和71%。处理方式不同,A549细胞收缩程度不同。
2.电镜图像给出的“bar”尺的长度与真实长度有误差,相对误差可达3%左右；不同放大倍数“bar”尺的误差程度不同.
3.A549细胞其超微结构具有以下量化特点
(1)A549细胞胞质约占细胞体积的72%,细胞核约占细胞体积的28%；以胞核为参照空间,核仁约占8%,染色质占92%,而染色质中76.9%是以常染色质的形式出现,异染色质约占23.1%；线粒体约占胞质的4.6%、板层小体约占胞质的2.5%、溶酶体和囊泡各约占胞质的1.39%和3.90%。
(2)平均一个A549细胞内约含501个线粒体,其表面积总量为1001.67μm2；约含950个板层小体,其表面积总量为428.68μm2；约含215个溶酶体,其表面积总量为212.04μm2；约含17409个囊泡,其表面积总量为1396.2μm2。
4.A549细胞和HBE细胞光镜下形态有差异,扫描电镜和透射电镜观察表明两组细胞形态相似。A549细胞核及核仁的形态与HBE细胞核及核仁的形态相比,核的平均偏离度为56.43%,核仁的平均偏离度为31.46%。
创新之处
1.首次用体视学的方法从量化角度及三维结构水平对A549细胞的超微结构进行了定量研究,A549细胞内胞质体积约占细胞体积的72%,细胞核体积约占细胞体积的28%；以胞核为参照空间,核仁约占8%,染色质占92%,而染色质中76.9%是以常染色质的形式出现,异染色质约占23.1%；线粒体约占胞质的4.6%、板层小体约占胞质的2.5%、溶酶体和囊泡各约占胞质的1.39%和3.90%。平均一个A549细胞内约含501个线粒体,其表面积总量为1001.67μm2；约含950个板层小体,其表面积总量为428.68μm2；约含215个溶酶体,其表面积总量为212.04μm2；约含17409个囊泡,其表面积总量为1396.2μm2。
2.首次以未固定、未脱水之A549细胞等渗重悬之平均直径为基准,测试其经过涂片HE染色、涂片巴氏染色和石蜡包埋3μm切片HE染色及透射电镜制样半薄(3μm)切片HE染色后的细胞收缩系数,发现A549细胞经过涂片HE染色、涂片巴氏染色和石蜡包埋3μm切片HE染色及透射电镜制样半薄(3μm)切片HE染色后会明显收缩,收缩系数分别为0.66、0.67、0.69和0.71；处理方式不同,A549细胞收缩程度不同,为光镜及电镜下对该细胞进行细胞及亚细胞的定量研究奠定了基础。
3.对电镜图像所给“bar”尺的真实长度进行测试,发现电镜图像“bar”尺所标的长度与其真实长度间会存在误差且不同放大倍数其误差程度不等。
4.应用体视学的原理和方法首次定量比较了A549细胞与HBE细胞核及核仁的差异,结果表明：A549细胞与HBE细胞相比,其核的平均偏离度为56.43%,核仁的平均偏离度为31.46%。
Background and objective
A549 human lung adenocarcinoma cell line, derived from human alveolar cell carcinoma, has a multi-containing body within cytoplasm.These body is typical in the lung alveolar typeⅡepithelial cells, so A549 cells were widely used both for diagnosis and treatment of lung cancer research, but also for the development and differentiation of lung epithelial cells in scientific research.According to Pubmed, by the now, more than 6300 articles using A549 cells were reported. These documents mainly focused on the functional studies of A549 cells.
However, it was well known that the changes of cell morphology was the basis of their function and in turn the changes of cell function can also affect morphology of cell.To increase understanding of cell function must be dependent on the knowledge and understanding of the morphology and structure of cell.
Morphological description was the best methods in the research biological morphology. Although a lot of questions can be resolved, there are a lot of shortcomings.Morphological description was subjective. We know that both the description of the "microscopic seen" and the ultra realistic under the optical or scanning or transmission electron microscope reflecting the structure and changes about morphology, which are limited to two-dimensional level and have no number of structure. Sometimes conclusions were completely wrong because of a variety of illusion such as Zoller Ebbinghaus illusion etc. For example, a circular in the two dimension structure does not mean necessarily that their three-dimension structure was the sphere. It may be a sphere, oval,round or irregular tubular structures.The structural view of two-dimension shape only displayed the various profiles of three-dimensional structure. The structural numberical view of two-dimensional picture only show a part of three-dimensional structure. Some small structure can not be found in two-dimension image. Thus the morphology of cell seen from two-dimension images such as the shape, size, number of the various structures will change. The simple description of findings from optical or electron microscope were very comprehensive or incomplete.
Stereology is the study off three-dimensional objects through the interpretation of two dimensional images.This is useful not only because it allows us to study the structure of entire cells and tissues based on thin sections or photomicrographs of sections, but also because it allows us to study this structural quantitatively. For example, the volume density of mitochondria could reflect the relative volume of mitochondria; the numberial density of mitochondrial can reflect the number of mitochondria; the surface density of mitochondrial membrane can reflect the mitochondrial membrane surface area; the mean distance of mitochondria can reflect the average space between mitochondria and mitochondria or reflect that the average distance between mitochondria and mitochondria are dense or sparse. Morfurther, the three-dimension structural features of nuclear, various organelles or inclusions etc. can been expressed using stereological parameters.
In present study, the morphological characteristics of A549 cells, nuclei,nucleoli, euchromatin, heterochromatin, mitochondria, lysosomes, lamellar body and vesicles were quantified from the images of light microscopy and electron microscopy by the application of stereological principles and methods using image analysis software Image-Pro Plus 6.0.The normal morphology parameter of cell,nucleus and other ultrastructure were established.The features of A549 cells were obtained from the three-dimension structural information.In the course of the study, we calibration some affect factors of the parameters such as cell contraction and calibration of test system.
Methods
1.Contraction coefficient of A549 cells in preparation for light and electron microscopy sample
(1)A549 cells were treated by resuspending in isotonic solution, smearing and Papanicolaou staining, smearing and HE staining, paraffin-embedded section and HE staining, transmission electron microscopy sample and semi-thin section and HE staining. A549 cells images were obtained through the inverted and light microscopy.
(2)Cells images were tested by application of Image-Pro Plus analysis software. The average diameter and coefficient of contraction of cells were calculated according to the stereological formula. 2.The analysis on the length error of "bar"on the electron microscopy images.
(1)Calibrate the test software with a grating replica and measure the actual length of the "bar" of 10μm,5μm and 2μm on the electron microscopy images of 3,500 times,8,000 times and 15,000 times by calibrated scale in test software respectively;
(2)calculate the absolute and relative error of the lengthe of the "bar" on images to the actual length of the "bar". 3.The analysis on the morphological characteristics of A549 cells ultrastructure
(1)A549 cells were observed under transmission electron microscopy and the various cell structures were identified;
(2) The morphological characteristics of A549 cells ultrastructure was tested by applications of the stereological principles and methods using Image-Pro Plus 6.0 image analysis software to design grid test systems.The stereological parameters included the nuclear volume density(VVn), the cytoplasm volume density(VVcyt),the nucleus surface density(SVn), the euchromatin volume density(VVeuc), the heterochrom-atin volume density(VVhet), the nucleolus volume density(VVnu), the nucleolus surface density(5Vnu), the mitochondrial density(VVmi), the mitochondrial surface density(SVmi), the mitochondrial numerical density(NVmi), the lysosome volume density(Vlys), the lysosome surface density(SVlys), the lysosome numerical density(NVlys),the lamellar body volume density(VVlam), the lamellar body surface area density(SVlam) and the lamellar body number density(NVlam).Absolute value included the average volume of cytoplasm(vcyt), the mean volume(vn) and the average surface area(sn) of nuclei, the mean volume(vn) and the average surface area(snu) of nucleolus; the average volume (vmi),the average surface area(smi) and number(nmi) of mitochondria; the average volume(vlys), the average surface area(slys) and the number(nlys) of lysosomes;the average volume(vlam), the average surface area(slam) and volume(nlam) of lamellar body. The density parameter of the nucleus and plasma were defined cells as reference space. The density parameter of the nucleolus, euchromatin and heterochromatin were defined cells as reference space The density parameter of the mitochondria, lysosomes,lamellar body were defined cytoplasm as reference space.
4.The comparative analysis of the morphological characteristics of nucleus and nucleolus between A549 cells and HBE cell
(1)The morphology of A549 and HBE cells were observed under optical microscopy and the inverted microscope. The morphological characteristics of nuclear and nucleolar were observed under electron microscopy.
(2) The area and perimeter of cells, nuclei and nucleoli were tested and the number of the nuclei and nucleus inside cells was counted using the mouse hook to describe the outline of the nucleus and nucleolus in each image by Image-Pro Plus 6.0 image analysis software. The parameters including the stereological parameters of nuclear: the volume density(VVn), the surface density(SVn), the surface area to volume ratio (RSVn), the numberical density(NVn), the mean volume(vn), the average surface area(sn) and the ratio cytoplasm(Rnp).The stereological parameters of nucleolus included the volume density(VVnu), the surface density(SVnu), the ratio of surface area to volume (RSVnu),the numberical density(NVnu),the average volume(vnu), the average surface area (snu) were measured and analized.
Results
1.The test results of contraction coefficient when A549 cell were prepared for the light and electron microscopy sample.
(1)The average cell diameter of the resuspensing A549 cell was 14.9302μm.The mean diameter of A549 cells was 9.8772μm,9.9775μm,10.2914μm and 10.5756μm by treated with smearing and HE staining, smearing and Pap staining, paraffin- embedded and HE staining, semi-thin section for TEM samples and HE staining respectively.
(2) The contraction coefficients of A549 cell treated with smearing and HE staining, smearing and Pap staining, paraffin-embedded and HE staining, semi-thin section for TEM samples and HE staining was 0.66,0.67,0.69 and 0.71 based on isotonic resuspended cells respectively.The method of treating cells including smearing and HE staining, smearing and Pap staining, paraffin-embedded and HE staining, semi-thin section for TEM samples and HE staining all can make A549 cells contract. The difference between the average diameter of isotonic resuspensing cells and other treated cells was signifcant(P<0.05).
2.The test results of the deviation of "bar" length on the TEM images
(1)The actual length of the "bar" given the length of 10μm on 3500 times TEM images was 10.3137μm ranging from 10.3065 to 10.3410μm and its coefficient of variation was 0.0011;The actual length of the "bar" given the length of 5μm on 8000 times TEM images was 5.1424μm ranging from 5.1360μm to 5.2079μm and its coefficient of variation was 0.0024;The actual length of the "bar" given the length of 2μm on 15000 times TEM images was 1.9265μm ranging from 1.9132μm to 1.9337μm and its coefficient of variation was 0.0046.It was all significant difference between the length of the "bar" on the images and the actual length of the "bar"(P=0.0 00).The difference between the actual length of "bar" and its length on 3,500 times, 8,000 times and 15,000 times TEM images was significant(P=0.000).
(2)The absolute error of "bar" on the electron microscope image of 3500 times was 0.3137μm ranging from 0.3065μm to 0.3410μm and its coefficient of variation was 0.0363;The absolute deviation of "bar" on TEM images of 8000 times was 0.1424μm ranging from 0.1360μm to 0.2079μm and its coefficient of variation was 0.0880; The absolute deviation of "bar" on TEM images of 15000 times was 0.0735μm ranging from 0.0663μm to 0.0868μm and its coefficient of variation was 0.1210.
(3) The relative error of "bar" on TEM images of 3500 times was 3.04% ranging from 2.97% to 3.30% and its coefficient of variation was 3.62%;The relative deviation of "bar" on TEM images of 8000 times was 2.77% ranging from 2.65% to 3.99% and its coefficient of variation was 8.30%;The relative error of "bar" on TEM images of 15000 times was 3.82% ranging from3.43% to 4.54% and its coefficient of variation was 12.56%.The overall difference of the relative error was significant on 3500 times,8000 times and 15,000 times TEM image(F=58.862,P=0.000).
3.The morphometric analysis on the ultrastructural characteristics of A549 cells
(1)The observation and identificati on ultrastructure of A549 cell under TEM
A large number of short microvilli were presented on A549 cell surface. Transmission electron microscopy revealed various cellular components such as mitochondria, lysosomes,microvilli, as well as the nucleus
(2) The quantitative characteristics of A549 cell ultrastructure
①The quantitative characteristics of A549 nuclear and plasma
The VVn was 0.2789,its 95 per cent confidence interval ranged from 0.2451 to 0.3129 and its coefficient of variation was 0.2191.The VVcyt was 0.7211,its 95 per cent confidence interval ranged from 0.6871 to 0.7549 and its coefficient of variation was 0.0847.The Rnp was 63%.The SVn was 0.1934μm-1,its 95 per cent confidence interval ranged from 0.1785 to 0.2075μm-1 and its coefficient of variation was 0.2004. The vn was 465.76μm3,its 95 per cent confidence interval ranged from 409.25 to 522.27μm3 and its coefficient of variation was 0.2091.The vcyt was 1204.2μm3,its 95 per cent confidence interval ranged from 1147.7 to 1260.7μm3 and its coefficient of variation was 0.0847.The sn was 322.92μm2, its 95 per cent confidence interval ranged from 298.76 to 347.08μm2 and its coefficient of variation was 0.2004.
②The quantitative characteristics of A549 nucleoclus,euchromatin, hetero-chromatin
The VVnu was 0.0761, its 95 per cent confidence interval ranged from 0.0739 to 0.0781 and its coefficient of variation was 0.0571.The SVnu was 0.1508μm-1,its 95 per cent confidence interval ranged from 0.1424 to 0.1596μm-1 and its coefficient of variation was 0.1518.The vnu was 35.623μm3,its 95 per cent confidence interval ranged from 34.497 to 36.749μm3 and its coefficient of variation was 0.0571.The snu was 70.517μm2, its 95 per cent confidence interval ranged from 66.521 to 74.513μm2 and its coefficient of variation was 0.1518.The VVeuc was 0.7225,its 95 per cent confidence interval ranged from 0.7097 to 0.7363 and its coefficient of variation was 0.0372.The veuc was 337.85μm3,its 95 per cent confidence interval ranged from 331.73 to 343.97μm3 and its coefficient of va riation was 0.0372.The VVhet was 0.2012, its 95 per cent confidence interval ranged from 0.1883 to 0.2137 and its coefficient of variation was 0.1164. The vhet was 94.114μm3, its 95 per cent confidence interval ranged from 88.047 to 100.18μm3 and its coefficient of variation was 0.1164.
③The quantitative characteristics of A549 cell mitochondria
The Vvmi was 0.0460, its 95 per cent confidence interval ranged from 0.0443 to 0.0477 and its coefficient of variation was 0.0883.The SVmi was 0.5988μm-1,its 95 per cent confidence interval ranged from 0.5045 to 0.6935μm-1 and its coefficient of variation was 0.5638.The NVmi was 0.3003μm-3,its 95 per cent confidence interval ranged from 0.2296 to 0.3704μm-3 and its coefficient of variation was 0.4977.The vmi was 76.823μm3,its 95 per cent confidence interval ranged from 58.882 to 64.938μm3 and its coefficient of variation was 0.0883.The smi was 1001.67μm, its 95 per cent confidence interval ranged from 888.09 to 1115.3μm2 and its coefficient of variation was 0.5683.The nmi was 501,its 95 per cent confidence interval ranged from 384 to 618 and its coefficient of variation was 0.4977.
④The quantitative characteristics of A549 cell lysosomes, lamellar bodies and vesicles
The VVlam was 0.0250, its 95 per cent confidence interval ranged from 0.0162 to 0.0338 and its coefficient of variation was 0.6242.The VVlys was 0.1290, its 95 per cent confidence interval ranged from 0.0091 to 0.0119 and its coefficient of variation was 0.5177.The VVves was 0.0390, its 95 per cent confidence interval ranged from 0.0397 to 0.0401 and its coefficient of variation was 0.0466.The SVlam was 0.3565μm-1,its 95 per cent confidence interval ranged from 0.2715 to 0.4425μm-1 and its coefficient of variation was 0.6432.The SVlys was 0.1763μm-1,its 95 per cent confidence interval ranged from 0.0987 to 0.2533μm-1 and its coefficient of variation was1.1744. The SVves was 1.1612μm-1,its 95 per cent confidence interval ranged from 1.0485 to 1.2735μm-1 and its coefficient of variation was 0.2591.The NVlam was 0.5691μm-3,its 95 per cent confidence interval ranged from 0.4337 to 0.7043μm-3 and its coefficient of variation was 0.7127.The Nvlys was 0.1289μm-3, its 95 per cent confidence interval ranged from 0.0874 to 0.1706μm-3 and its coefficient of variation was 0.4624.The NVves was 10.424μm-3,its 95 per cent confidence interval ranged from 8.7206 to 12.274μm-3 and its coefficient of variation was 0.3178.The vlam was 41.763μm3,its 95 per cent confidence interval ranged from 62.384 to 91.257μm3 and its coefficient of variation was 0.6242. The vlys was 21.694μm3,its 95 per cent confidence interval ranged from 15.474 to 27.914μm3 and its coefficient of variation was 0.5177.The vves was 65.241μm3,its 95 per cent confidence interval ranged from 63.774 to 66.708μm3 and its coefficient of variation was 0.0406.The slam was 428.68μm2,its 95 per cent confidence interval ranged from 325.73 to 531.63μm2 and its coefficient of variation was 0.6432.The slys was 212.04μm2, its 95 per cent confidence interval ranged from 119.06 to 305.02μm2 and its coefficient of variation was 1.1744. The sves was 1396.2μm2, its 95 per cent confidence interval ranged from 1261.1 to 1531.3μm2 and its coefficient of variation was 0.2591.The nlam was 950, its 95 per cent confidence interval ranged from 724 to 1175 and its coefficient of variation was 0.7127.The nlys was 215,its 95 per cent confidence interval ranged from 146 to 284 and its coefficient of variation was 0.4624. The nves was 17,409, its 95 per cent confidence interval ranged from 14,564 to 20,253 and its coefficient of variation was 0.3178.
4. The comparative results of the morphological characteristics between nucleus and nucleolus of A549 and HBE cell s.
(1)The observation under cells seeding and HE staining and inverted microscopy.
A549 and HBE cells have visible difference in size and appearance. A549 cells was spindle-shaped and mainly scattered. HBE cells like polygonal epithelioid cells.
(2) The morphology of A549 and HBE cells under SEM and TEM.
Scanning electron microscope:The microvilla are presented on the surface of HBE cells and A549 cells. The irregular nuclei can be found under TEM observation of nuclei and nucleoli.Some nucleus contained two or more nucleolus. There are inclusions within both HBE cells and cells nucleus.
(3) The quantitative characteristics of A549 cells and HBE cells nucleus and nucleolus at three-dimensional level.
Based on the morphological parameters of HBE nucleus and nucleoli, the negative deviation of the volume density, nuclear surface area density, nuclear numerical density and the nuclear ratio surface to volume of A549 nuclear was 21.14%,27.96%,32.87% and 50% respectively. The positive deviation of the nuclear average volume, the average surface area of nuclear and the ratio of nuclear to cytoplasm of A549 were 146.98% and 59.65% and 12.90%.The negative deviation of A549 nucleolar volume density, surface area density, number density and the nucleolar ratio surface to volume was 7.81%,33.63%,36.64%and 43.68%.The positive deviation of A549 nucleolar average volume, the average surface area was 53.49% and 13.48% respectively.
Conclusion
1.The contraction level of A549 cells after treated with smearing and HE staining, smearing and Pap staining, paraffin-embedded and HE staining, transmission electron microscopy sample preparation and HE staining processing were large:66%,67%,69%, 71% respectively. The average diameter of A549 cell in different method are different.
2.There was the error between the length of "bar" on TEM image and the actual length of "bar".The error of "bar" was difference when magnification was different. The relative error of "bar" on the 3500 times,8000 times and 15,000 times images were 3.04%,2.77% and 3.82% respectively. The error of the "bar" on images of 15,000 times,8,000 times and 3,500 times was significantly difference.
3.The quantitative characteristics of A549 cell ultrastructure
(1)The individual A549 cell contained 72% cytoplasm and 28% nucleus.The nucleus contained about 8% nucleolus and about 92% chromatin,of which about 76.9% appear in the euchromatin form. About 4.6% of cytoplasm was made up of mitochondria. The lamellar body space amounted to 2.5% of cytoplasm, while autolysosomes and vesicles contributed 1.39% and 3.9% of cytoplasm respectively. Total mentioned organelles occupied about 15% of cytoplasm.
(2) In invidual A549 cell total surface area of mitochondria was 1001.67μm2 and the number of mitochondria was about 501.Total surface area of lamellar body was 428.68μm2 and the number of lamellar body was 950. Total surface area of lysosome was 212.04μm2 and the number of lysosome was approximately 215.Total surface area of vesicles was 1396.2μm2 and the number of vesicles was about 17409. 4. There was deviation between the morphology of A549 nucleus and nucleolus and HBE nucleus and nucleolus.The average degree of deviation of A549 nuclear was 56.43%.The average degree of deviation of A549 nucleolar was 31.46%.
New points
1.Through stereological studies on the ultrastructure of A549 cell we found the individual A549 cell contained 72% cytoplasm and 28% nucleus. The nucleus contained about 8% nucleolus and about 92% chromatin, of which about 76.9% appear in the euchromatin form.About 4.6% of cytoplasm was made up of mitochondria. The lamellar body space amounted to 2.5%of cytoplasm, while autolysosomes and vesicles contributed 1.39% and 3.9% of cytoplasm respectively. Total mentioned organelles occupied about 15% of cytoplasm.In invidual A549 cell total surface area of mitochondria was 1001.67μm2 and the number of mitochondria was about 501.Total surface area of lamellar body was 428.68μm2 and the number of lamellar body was 950.Total surface area of lysosome was 212.04μm2 and the number of lysosome was approximately 215.Total surface area of vesicles was 1396.2μm2 and the number of vesicles was about 17409.
2.The contraction level of A549 cells after treated with smearing and HE staining,smearing and Pap staining, paraffin-embedded and HE staining, transmission electron microscopy sample preparation and HE staining processing were large: 66%,67%,69%,71% respectively. The average diameter of A549 cell in different method are different.
3.There was the error between the length of "bar" on TEM image and the actual length of "bar".The error of "bar" was difference when magnification was different. The relative error of "bar" on the 3500 times,8000 times and 15,000 times images were 3.04%,2.77% and 3.82% respectively. The error of the "bar" on images of 15,000 times,8,000 times and 3,500 times was significantly difference.
4.There was deviation between the morphology of A549 nucleus and nucleolus and HBE nucleus and nucleolus.The average degree of deviation of A549 nuclear was 56.43%.The average degree of deviation of A549 nucleolar was 31.46%.
A549细胞来源于人肺泡细胞癌,由Giard D J等于1973年建立。该细胞既具有肺癌恶性肿瘤细胞的特性,同时又具有肺泡Ⅱ型上皮细胞的特性和表型。故在研究中A549细胞被广泛用于肺癌的发生、发展、诊断及治疗研究,也被用于正常人肺泡Ⅱ型上皮细胞的发生、分化及其结构和功能的研究。根据Pubmed查询,到目前为止,使用A549细胞进行研究的英文报道达6200余篇；CNKI收录的利用A549细胞进行研究的中文文献多达4600余篇,且中英文献数均逐年呈倍数级上升。然而,这些报道主要集中于A549细胞功能方面的研究。
众所周知,细胞形态变化是其功能变化的基础,而细胞功能方面的变化又将影响细胞形态结构。要想提高对细胞功能的理解和认识必须深化对细胞形态和结构的认识。以往对形态的认识和研究多局限于镜下的定性观察。然而,对A549细胞的形态结构研究,无论是光学显微镜之“镜下所见”,还是扫描电镜抑或透射电镜下的超微结构观察,采用的均为经典的视觉描述来反映细胞的形态结构及其变化,且都局限于二维水平,缺乏形态结构的量化概念,缺乏三维结构水平的定量研究。而且由于各种视觉错觉的作用,如佐勒错觉和埃宾豪斯错觉等,有时甚至会得出完全错误的结论。从结构形态来看二维图像也只是显示三维结构的各种剖面,例如对于二维切片中的圆形结构并不一定意味着其三维结构为球体,它可能是球体,也可能是椭圆、圆管状或不规则结构等。从结构数量来看,二维图像一般只能显示三维结构的一部分,有些结构数目少,分布不均匀时,在二维图像中往往看不到。因此,二维图像上所见的细胞形态,诸如各种结构的形状、大小、多少都发生了变化,单纯描述镜下所见并不全面,也不完善。
体视学是由二维结构信息定量推论三维结构信息的介于形态学与数学之间的新兴学科。应用体视学原理最终可测得细胞的三维结构参数,使人们能从三维水平定量地阐明细胞的结构。例如,细胞中线粒体的体密度能反映出线粒体相对体积大小,线粒体数密度反映了线粒体数量多少,线粒体内膜表面积密度可反映线粒体内膜表面积大小,线粒体的平均自由程则反映出线粒体与线粒体之间的空间平均距离,或者反映了线粒体是密集还是稀少,而线粒体的曲度、球度、轴比等则从不同方面反映了线粒体的形态等。其他诸如细胞、核、各种细胞器及细胞中的包含物等的三维结构特征都可用相应的体视学参数来定量描述。
本研究利用光镜与电镜拍摄细胞的图像,采用图像分析软件Image-Pro Plus6.0对A549细胞的图像进行分析,并应用体视学的原理和方法,对A549细胞及其细胞核内的核仁、常染色质、异染色质以及细胞质内的线粒体、溶酶体、板层小体及囊泡等超微结构的三维形态特征进行量化,从三维水平定量揭示A549细胞超微结构的特点。在研究过程中,我们还对影响参数结果的因素进行了校正如细胞收缩的校正及测试系统的标定等。
方法
A549细胞株和HBE细胞株由病理教研室液氮保存。
1.A549细胞光镜及电镜制样收缩系数测试
(1)分别用等渗重悬、涂片HE染色、涂片巴氏染色、石蜡包埋3μm切片HE染色及透射电镜制样半薄(3μm)切片HE染色5种常规方法处理细胞；
(2)用Image-Pro Plus 6.0图像分析软件测试每组细胞的平均直径,然后以A549培养之等渗重悬细胞之平均直径为基准计算细胞的收缩系数。
2.电镜图像“bar”尺长度的误差分析
(1)用光栅复型标定测试软件并用软件按标定后的标准尺度测量3500倍、8000倍和15000倍电镜图像上对应给出的10μm、5μm和2μm“bar”尺的真实长度,用测得的“bar”尺真实长度与图像所标的长度进行比较分析；
(2)计算电镜图像“bar”尺所给的长度与其真实长度间的绝对误差和相对误差,比较不同放大倍数图像上“bar”尺所给的长度与其真实长度间的相对误差差异程度。3.A549细胞超微结构体视学研究
(1)透射电镜下观察A549细胞内细胞器的结构并予辨认；
(2)用Image-Pro Plus 6.0图像分析软件设计测试系统,应用体视学原理和方法,测试A549细胞超微结构的体视学参数包括：细胞核体密度(VVn)、细胞浆体密度(VVcyt)、细胞核表面积密度(SVn)、核内常染色质体密度(VVeuc)、异染色质体密度(VVhet)、核仁体密度(VVnu)、核仁表面积密度(SVnu)、线粒体体密度(VVmi)、线粒体表面积密度(SVmi)、线粒体数密度(NVmi)；溶酶体体密度(VVlys)、溶酶体表面积密度(SVlys)、溶酶体数密度；板层小体体密度(Vlam)、板层小体表面积密度(SVlam)、板层小体数密度(VVlam)；囊泡体密度(VVves)、囊泡表面积密度(SVves)、囊泡数密度(NVves)；细胞浆平均体积(vcyt)、细胞核平均体积(vn)及平均表面积(sn)；核仁平均体积(vn)及平均表面积(snu)；线粒体的平均体积(vmi)、平均表面积(smi)及数量(nmi);溶酶体的平均体积(vlys)、平均表面积(slys)及数量(nlys)；板层小体的平均体积(nlam)、平均表面积(slam)及数量(nlam)；囊泡的平均体积(vves)、平均表面积(sves)及数量(nves)。上述参数中的细胞浆平均体积(vcyt)、细胞核平均体积(vn)及平均表面积·(sn),核仁平均体积(vn)及平均表面积(snu),线粒体的平均体积(vmi)、平均表面积(smi)及数量(nmi),溶酶体的平均体积(vlys)、平均表面积(slys)及数量(nlys),板层小体的平均体积(nlam)、平均表面积(slam)及数量(nlam)及囊泡的平均体积(vves)、平均表面积(sves)及数量(nves)是指一个A549细胞内对应细胞器参数的绝对含量。细胞核及浆的密度参数以细胞为参照空间,核仁、常染色质及异染色质的密度参数以细胞核为参照空间,线粒体、溶酶体、板层小体及囊泡结构的密度参数以细胞浆为参照空间。
4.A549细胞和HBE细胞形态结构的差异分析
(1)光学显微镜、倒置显微镜、扫描电镜及透射电镜下观察A549细胞和HBE细胞的形态特点；
(2)通过Image-Pro Plus 6.0图像分析软件鼠标勾描每幅图像中的细胞、细胞核和核仁的轮廓,分别测试细胞、细胞核及核仁的面积和周长,并计数细胞内的胞核数及核内的核仁数,以细胞为参照空间计算HBE细胞和A549细胞核及核仁形态的体视学参数：包括细胞核体密度(VVn)、细胞核表面积密度(SVn)、细胞核表面积与体积比(RSVn)、细胞核数密度(NVn)、细胞核平均体积(vn)、细胞核平均表面积(Sn)和核浆比(Rnp)；细胞核仁的体视学参数包括核仁体密度(VVnu)、核仁表面积密度(SVnu)、核仁表面积与体积比(RSVnu)、核仁数密度(NVnu)、核仁平均体积(Vnu)、核仁平均表面积(Snu)共13种体视学参数的数值。
(3)以HBE细胞核及核仁形态参数值为基准,计算A549细胞核及核仁形态的正负偏离度。
结果
1.A549细胞光镜及电镜制样的收缩系数测试
(1)A549细胞平均直径的测试结果
等渗液重悬A549细胞的平均直径为14.9302μm,范围在10.0248-19.3552μm之间,变异系数为0.0998；涂片HE染色A549细胞的平均直径为9.8775μm,范围在7.5651-14.4611μm之间,变异系数为0.1009；涂片巴氏染色A549细胞的平均直径为9.9772μm,范围在7.6697-14.5422μm之间,变异系数为0.1071；石蜡包埋3μm厚切片HE染色A549细胞的平均直径为10.2914μm,范围在6.0952-16.1046μm之间,变异系数为0.1419；透射电镜半薄切片HE染色A549细胞的平均直径为10.5857μm,范围在3.6853-20.3462μm之间,变异系数为0.2653。
(2)A549细胞收缩系数的计算结果
以等渗重悬细胞的平均直径为基准,涂片HE染色、涂片巴氏染色、石蜡包埋3μm厚切片和透射电镜制样环氧树脂包埋3μm厚切片HE染色后细胞的收缩系数分别为：0.66、0.67、0.69和0.71。经过涂片HE染色、涂片巴氏染色、石蜡包埋切片HE染色和电镜制样半薄切片HE染色后A549细胞均有较明显的收缩,其平均直径与等渗重悬的A549细胞的平均直径比较差异均有显著性(P=0.000)。
2.电镜图像“bar”尺长度误差的测试
(1)电镜图像上“bar”尺的真实长度测试结果
3500倍图像长度为10μm“bar”尺的真实长度均值为10.3137μm,范围在10.3065-10.3410μm之间,变异系数为0.0011；8000倍图像长度为5μm“bar”尺的真实长度均值为5.1424μm,范围在5.1360-5.2079μm之间,变异系数为0.0024；15000倍长度为2μm“bar”尺的真实长度均值为1.9265μm,范围在1.9132-1.9337μm之间,变异系数为0.0046。3500倍、8000倍和15000倍电镜图像上“bar”尺的长度与其真实长度间的比较差异均有显著性(P=0.000)。
(2)电镜图像上“bar”尺长度与其真实长度间的绝对误差计算结果
3500倍图像“bar”尺长度与其真实长度间的绝对误差均值为0.3137μm,范围在0.3065-0.3410μm之间,变异系数为0.0363；8000倍图像“bar”尺长度与其真实长度间的绝对误差均值为0.1424μm,范围在0.1360-0.2079μm之间,变异系数为0.0880；15000倍图像“bar”尺长度与其真实长度间的绝对误差均值为0.0735μm,范围在0.0663-0.0868 u m之间,变异系数为0.1210。
(3)电镜图像上“bar”尺长度与其真实长度间的相对误差计算结果
3500倍图像“bar”尺长度与其真实长度间的相对误差均值为3.04%,范围在2.97%-3.30%之间,变异系数为3.62%；8000倍图像“bar”尺长度与其真实长度间的相对误差均值为2.77%,范围在2.65%-3.99%之间,变异系数为8.30%；15000倍图像“bar”尺长度与其真实长度间的相对误差均值为3.82%,范围在3.43%-4.54%之间,变异系数为12.56%。3500倍、8000倍和15000倍电镜图像“bar”尺长度与其真实长度间的相对误差三种不同放大倍数整体比较差异有显著性(F=58.862,P=0.000)。3500倍、8000倍和15000倍电镜图像上“bar”尺长度与其真实长度间的相对误差两两比较差异均有显著性(P=0.000)。
3.A549细胞三维超微结构的形态分析
(1)A549细胞透射电镜下超微结构观察
A549细胞表面有大量的短小微绒毛。细胞质丰富,内有各种细胞器如线粒体、溶酶体、内质网、高尔基复合体、板层小体、粘液空泡和微囊等。线粒体在细胞中分布不均一,呈极性分布；溶酶体大部分属于自噬溶酶体,由单位膜包绕,其内含有残余的细胞器；板层小体为圆形或卵圆形,内部有呈平行或螺纹状排列的板状结构；微囊的腔面见微绒毛,腔内有不同电子密度的分泌物；粘液空泡往往几个或一群密集分布,其电子密度较低。局部区域内质网、高尔基复合体结构丰富。细胞核大,核形不规则,可见极不规则核；核膜皱褶多；染色质细密,常染色质占大部分；核内可见一个或多个核仁。观察、辨认细胞电镜图像发现板层小体和微囊两种超微结构,可作为鉴定A549细胞的佐证之一
(2)A549细胞超微结构形态的量化特点
①A549细胞核及浆形态的量化特点
VVn均值为0.2789,其95%的可信区间在0.2451-0.3129之间,变异系数为0.2191；VVcyt均值为0.7211,其95%的可信区间在0.6871-0.7549之间,变异系数为0.0847；Rnp为0.63；SVn均值为0.1934μm-1,其95%的可信区间在0.1785-0.2075μm-1之间,变异系数为0.2004；RSVn为0.67；vn均值为465.76μm3,其95%的可信区间在409.25-522.27μm3之间,变异系数为0.2091；vcyt均值为1204.2μm3,其95%的可信区间在1147.7-1260.7μm3之间,变异系数为0.0847；sn均值为322.92μm2,其95%的可信区间在298.76-347.08μm2之间,变异系数为0.2004。
②A549细胞核内核仁、常染色质及异染色质形态的量化特点
VVnu。均值为0.0761,其95%的可信区间在0.0739-0.0781之间,变异系数为0.0571；SVnu均值为0.1508μm-1,其95%的可信区间在0.1424-0.1596μm-1之间,变异系数为0.1518,因此A549细胞核仁的表面积与体积比为1.98。vnu均值为35.623μm3,其95%的可信区间在34.497-36.749μm3之间,变异系数为0.0571；snu均值为70.517μm2,其95%的可信区间在66.521-74.513μm2之间,变异系数为0.1518。VVeuc均值为0.7225,其95%的可信区间在0.7097-0.7363之间,变异系数为0.0372；Veuc均值为337.85μm3,其95%的可信区间在331.73-343.97μm3之间,变异系数为0.0372。VVher均值为0.2012,其95%的可信区间在0.1883-0.2137之间,变异系数为0.1164；Vhet均值为94.114μm3,其95%的可信区间在88.047-100.18μm-3之间,变异系数为0.1164。
③A549细胞线粒体形态的量化特点
VVmi均值为0.0460,其95%的可信区间在0.0443-0.0477之间,变异系数为0.0883；SVmi,均值为0.5988μm3,其95%的可信区间在0.5045-0.6935μm-1之间,变异系数为0.5638；NVmi均值为0.3003μm-3,其95%的可信区间在0.2296-0.3704μm-3之间,变异系数为0.4977。vmi均值为61.910μm3,其95%的可信区间在58.882-64.938μm3之间,变异系数为0.0883；smi均值为1001.67μm2,其95%的可信区间在888.09-1115.3μm2之间,变异系数为0.5683；一个A549细胞含nmi约501个,其95%的可信区间在384-618个之间,变异系数为0.4977。
④A549细胞溶酶体、板层小体和囊泡形态的量化特点
VVlan均值为0.0250,其95%的可信区间在0.0162-0.0338之间,变异系数为0.6242；VVlys均值为0.0129,其95%的可信区间在0.0091-0.0119之间,变异系数为0.5177；VVves均值为0.0390,其95%的可信区间在0.0397-0.0401之间,变异系数为0.0466。SVlam均值为0.3565μm-1,其95%的可信区间在0.2715-0.4425μm-1之间,变异系数为0.6432；SVlys均值为0.1763μm-1,其95%的可信区间在0.0987-0.2533μm-1之间,变异系数为1.1744：SVves均值为1.1612μm-1,其95%的可信区间在1.0485-1.2735μm-1之间,变异系数为0.2591。NVlam均值为0.5691μm-3,其95%的可信区间在0.4337-0.7043μm-3。之间,变异系数为0.7127；NVlys均值为0.1289μm-3,其95%的可信区间为0.0874-0.1706μm-3之间,变异系数为0.4624；NVves均值为10.424μm-3,其95%的可信区间在8.7206-12.274μm-3之间,变异系数为0.3178；vlam值为76.820μm3,其95%的可信区间在62.384-91.257μm-3之间,变异系数为0.6242；vlys均值为21.694μm-3,其95%的可信区间在15.474-27.914μm-3之间,变异系数为0.5177；vves均值为65.241μm3,其95%的可信区间在63.774-66.708μm3之间,变异系数为0.0406；slam均值为428.68μm2,其95%的可信区间在325.73-531.63μm2之间,变异系数为0.6432；slys均值为212.04μm2,其95%的可信区间在119.06-305.02μm2之间,变异系数为1.1744；sves均值为1396.2μm2,其95%的可信区间在1261.1-1531.3μm2之间,变异系数为0.2591；一个A549细胞内,nlam约含950个,其95%的可信区间在724-1175个之间,变异系数为0.7127.nlys约含215个,其95%的可信区在146-284个之间,变异系数为0.4624；nves约含17409个,其95%的可信区间在14564-20253个之间,变异系数为0.3178.
4.A549和HBE细胞形态结构差异分析
(1)倒置显微镜和细胞爬片HE染色观察结果
A549细胞和HBE细胞均为大小不一、形态多样。A549以梭形、多角形细胞为主,散在、单层排列,HBE细胞以多边形上皮样细胞为主,梭形细胞只占极少数,镶嵌排列。HBE细胞形态相对A549较一致。
(2)扫描电镜及透射电镜下A549细胞和HBE细胞形态观察结果
扫描电镜下：HBE细胞和A549细胞的表面可见细胞皱褶、突起和细小均匀分布的纤毛。透射电镜观察,HBE细胞和A549细胞中可见不规则细胞核,其内见一个或多个核仁。
(3)A549细胞及HBE细胞核及核仁形态差异定量分析结果
以HBE细胞核及核仁的形态参数为基准,A549细胞核体密度、核表面积密度、核数密度和核表面积与体积比的负偏离度分别为21.14%、27.96%、50%和32.87%,A549细胞核平均体积、核平均表面积和核浆比的正偏离度分别为146.98%、59.65%和12.90%,A549细胞核仁体积密度、核仁表面积密度、核仁数密度及核仁表面积与体积比的负偏离度为7.81%、33.63%、43.68%和36.64%,A549细胞核仁的平均体积、核仁平均表面积的正偏离度分别为53.49%和13.48%。
结论
1.A549细胞经过涂片HE染色、涂片巴氏染色和石蜡包埋3μm切片HE染色及透射电镜制样半薄(3μm)切片HE染色后会明显收缩,收缩系数分别为：66%、67%、69%和71%。处理方式不同,A549细胞收缩程度不同。
2.电镜图像给出的“bar”尺的长度与真实长度有误差,相对误差可达3%左右；不同放大倍数“bar”尺的误差程度不同.
3.A549细胞其超微结构具有以下量化特点
(1)A549细胞胞质约占细胞体积的72%,细胞核约占细胞体积的28%；以胞核为参照空间,核仁约占8%,染色质占92%,而染色质中76.9%是以常染色质的形式出现,异染色质约占23.1%；线粒体约占胞质的4.6%、板层小体约占胞质的2.5%、溶酶体和囊泡各约占胞质的1.39%和3.90%。
(2)平均一个A549细胞内约含501个线粒体,其表面积总量为1001.67μm2；约含950个板层小体,其表面积总量为428.68μm2；约含215个溶酶体,其表面积总量为212.04μm2；约含17409个囊泡,其表面积总量为1396.2μm2。
4.A549细胞和HBE细胞光镜下形态有差异,扫描电镜和透射电镜观察表明两组细胞形态相似。A549细胞核及核仁的形态与HBE细胞核及核仁的形态相比,核的平均偏离度为56.43%,核仁的平均偏离度为31.46%。
创新之处
1.首次用体视学的方法从量化角度及三维结构水平对A549细胞的超微结构进行了定量研究,A549细胞内胞质体积约占细胞体积的72%,细胞核体积约占细胞体积的28%；以胞核为参照空间,核仁约占8%,染色质占92%,而染色质中76.9%是以常染色质的形式出现,异染色质约占23.1%；线粒体约占胞质的4.6%、板层小体约占胞质的2.5%、溶酶体和囊泡各约占胞质的1.39%和3.90%。平均一个A549细胞内约含501个线粒体,其表面积总量为1001.67μm2；约含950个板层小体,其表面积总量为428.68μm2；约含215个溶酶体,其表面积总量为212.04μm2；约含17409个囊泡,其表面积总量为1396.2μm2。
2.首次以未固定、未脱水之A549细胞等渗重悬之平均直径为基准,测试其经过涂片HE染色、涂片巴氏染色和石蜡包埋3μm切片HE染色及透射电镜制样半薄(3μm)切片HE染色后的细胞收缩系数,发现A549细胞经过涂片HE染色、涂片巴氏染色和石蜡包埋3μm切片HE染色及透射电镜制样半薄(3μm)切片HE染色后会明显收缩,收缩系数分别为0.66、0.67、0.69和0.71；处理方式不同,A549细胞收缩程度不同,为光镜及电镜下对该细胞进行细胞及亚细胞的定量研究奠定了基础。
3.对电镜图像所给“bar”尺的真实长度进行测试,发现电镜图像“bar”尺所标的长度与其真实长度间会存在误差且不同放大倍数其误差程度不等。
4.应用体视学的原理和方法首次定量比较了A549细胞与HBE细胞核及核仁的差异,结果表明：A549细胞与HBE细胞相比,其核的平均偏离度为56.43%,核仁的平均偏离度为31.46%。
Background and objective
A549 human lung adenocarcinoma cell line, derived from human alveolar cell carcinoma, has a multi-containing body within cytoplasm.These body is typical in the lung alveolar typeⅡepithelial cells, so A549 cells were widely used both for diagnosis and treatment of lung cancer research, but also for the development and differentiation of lung epithelial cells in scientific research.According to Pubmed, by the now, more than 6300 articles using A549 cells were reported. These documents mainly focused on the functional studies of A549 cells.
However, it was well known that the changes of cell morphology was the basis of their function and in turn the changes of cell function can also affect morphology of cell.To increase understanding of cell function must be dependent on the knowledge and understanding of the morphology and structure of cell.
Morphological description was the best methods in the research biological morphology. Although a lot of questions can be resolved, there are a lot of shortcomings.Morphological description was subjective. We know that both the description of the "microscopic seen" and the ultra realistic under the optical or scanning or transmission electron microscope reflecting the structure and changes about morphology, which are limited to two-dimensional level and have no number of structure. Sometimes conclusions were completely wrong because of a variety of illusion such as Zoller Ebbinghaus illusion etc. For example, a circular in the two dimension structure does not mean necessarily that their three-dimension structure was the sphere. It may be a sphere, oval,round or irregular tubular structures.The structural view of two-dimension shape only displayed the various profiles of three-dimensional structure. The structural numberical view of two-dimensional picture only show a part of three-dimensional structure. Some small structure can not be found in two-dimension image. Thus the morphology of cell seen from two-dimension images such as the shape, size, number of the various structures will change. The simple description of findings from optical or electron microscope were very comprehensive or incomplete.
Stereology is the study off three-dimensional objects through the interpretation of two dimensional images.This is useful not only because it allows us to study the structure of entire cells and tissues based on thin sections or photomicrographs of sections, but also because it allows us to study this structural quantitatively. For example, the volume density of mitochondria could reflect the relative volume of mitochondria; the numberial density of mitochondrial can reflect the number of mitochondria; the surface density of mitochondrial membrane can reflect the mitochondrial membrane surface area; the mean distance of mitochondria can reflect the average space between mitochondria and mitochondria or reflect that the average distance between mitochondria and mitochondria are dense or sparse. Morfurther, the three-dimension structural features of nuclear, various organelles or inclusions etc. can been expressed using stereological parameters.
In present study, the morphological characteristics of A549 cells, nuclei,nucleoli, euchromatin, heterochromatin, mitochondria, lysosomes, lamellar body and vesicles were quantified from the images of light microscopy and electron microscopy by the application of stereological principles and methods using image analysis software Image-Pro Plus 6.0.The normal morphology parameter of cell,nucleus and other ultrastructure were established.The features of A549 cells were obtained from the three-dimension structural information.In the course of the study, we calibration some affect factors of the parameters such as cell contraction and calibration of test system.
Methods
1.Contraction coefficient of A549 cells in preparation for light and electron microscopy sample
(1)A549 cells were treated by resuspending in isotonic solution, smearing and Papanicolaou staining, smearing and HE staining, paraffin-embedded section and HE staining, transmission electron microscopy sample and semi-thin section and HE staining. A549 cells images were obtained through the inverted and light microscopy.
(2)Cells images were tested by application of Image-Pro Plus analysis software. The average diameter and coefficient of contraction of cells were calculated according to the stereological formula. 2.The analysis on the length error of "bar"on the electron microscopy images.
(1)Calibrate the test software with a grating replica and measure the actual length of the "bar" of 10μm,5μm and 2μm on the electron microscopy images of 3,500 times,8,000 times and 15,000 times by calibrated scale in test software respectively;
(2)calculate the absolute and relative error of the lengthe of the "bar" on images to the actual length of the "bar". 3.The analysis on the morphological characteristics of A549 cells ultrastructure
(1)A549 cells were observed under transmission electron microscopy and the various cell structures were identified;
(2) The morphological characteristics of A549 cells ultrastructure was tested by applications of the stereological principles and methods using Image-Pro Plus 6.0 image analysis software to design grid test systems.The stereological parameters included the nuclear volume density(VVn), the cytoplasm volume density(VVcyt),the nucleus surface density(SVn), the euchromatin volume density(VVeuc), the heterochrom-atin volume density(VVhet), the nucleolus volume density(VVnu), the nucleolus surface density(5Vnu), the mitochondrial density(VVmi), the mitochondrial surface density(SVmi), the mitochondrial numerical density(NVmi), the lysosome volume density(Vlys), the lysosome surface density(SVlys), the lysosome numerical density(NVlys),the lamellar body volume density(VVlam), the lamellar body surface area density(SVlam) and the lamellar body number density(NVlam).Absolute value included the average volume of cytoplasm(vcyt), the mean volume(vn) and the average surface area(sn) of nuclei, the mean volume(vn) and the average surface area(snu) of nucleolus; the average volume (vmi),the average surface area(smi) and number(nmi) of mitochondria; the average volume(vlys), the average surface area(slys) and the number(nlys) of lysosomes;the average volume(vlam), the average surface area(slam) and volume(nlam) of lamellar body. The density parameter of the nucleus and plasma were defined cells as reference space. The density parameter of the nucleolus, euchromatin and heterochromatin were defined cells as reference space The density parameter of the mitochondria, lysosomes,lamellar body were defined cytoplasm as reference space.
4.The comparative analysis of the morphological characteristics of nucleus and nucleolus between A549 cells and HBE cell
(1)The morphology of A549 and HBE cells were observed under optical microscopy and the inverted microscope. The morphological characteristics of nuclear and nucleolar were observed under electron microscopy.
(2) The area and perimeter of cells, nuclei and nucleoli were tested and the number of the nuclei and nucleus inside cells was counted using the mouse hook to describe the outline of the nucleus and nucleolus in each image by Image-Pro Plus 6.0 image analysis software. The parameters including the stereological parameters of nuclear: the volume density(VVn), the surface density(SVn), the surface area to volume ratio (RSVn), the numberical density(NVn), the mean volume(vn), the average surface area(sn) and the ratio cytoplasm(Rnp).The stereological parameters of nucleolus included the volume density(VVnu), the surface density(SVnu), the ratio of surface area to volume (RSVnu),the numberical density(NVnu),the average volume(vnu), the average surface area (snu) were measured and analized.
Results
1.The test results of contraction coefficient when A549 cell were prepared for the light and electron microscopy sample.
(1)The average cell diameter of the resuspensing A549 cell was 14.9302μm.The mean diameter of A549 cells was 9.8772μm,9.9775μm,10.2914μm and 10.5756μm by treated with smearing and HE staining, smearing and Pap staining, paraffin- embedded and HE staining, semi-thin section for TEM samples and HE staining respectively.
(2) The contraction coefficients of A549 cell treated with smearing and HE staining, smearing and Pap staining, paraffin-embedded and HE staining, semi-thin section for TEM samples and HE staining was 0.66,0.67,0.69 and 0.71 based on isotonic resuspended cells respectively.The method of treating cells including smearing and HE staining, smearing and Pap staining, paraffin-embedded and HE staining, semi-thin section for TEM samples and HE staining all can make A549 cells contract. The difference between the average diameter of isotonic resuspensing cells and other treated cells was signifcant(P<0.05).
2.The test results of the deviation of "bar" length on the TEM images
(1)The actual length of the "bar" given the length of 10μm on 3500 times TEM images was 10.3137μm ranging from 10.3065 to 10.3410μm and its coefficient of variation was 0.0011;The actual length of the "bar" given the length of 5μm on 8000 times TEM images was 5.1424μm ranging from 5.1360μm to 5.2079μm and its coefficient of variation was 0.0024;The actual length of the "bar" given the length of 2μm on 15000 times TEM images was 1.9265μm ranging from 1.9132μm to 1.9337μm and its coefficient of variation was 0.0046.It was all significant difference between the length of the "bar" on the images and the actual length of the "bar"(P=0.0 00).The difference between the actual length of "bar" and its length on 3,500 times, 8,000 times and 15,000 times TEM images was significant(P=0.000).
(2)The absolute error of "bar" on the electron microscope image of 3500 times was 0.3137μm ranging from 0.3065μm to 0.3410μm and its coefficient of variation was 0.0363;The absolute deviation of "bar" on TEM images of 8000 times was 0.1424μm ranging from 0.1360μm to 0.2079μm and its coefficient of variation was 0.0880; The absolute deviation of "bar" on TEM images of 15000 times was 0.0735μm ranging from 0.0663μm to 0.0868μm and its coefficient of variation was 0.1210.
(3) The relative error of "bar" on TEM images of 3500 times was 3.04% ranging from 2.97% to 3.30% and its coefficient of variation was 3.62%;The relative deviation of "bar" on TEM images of 8000 times was 2.77% ranging from 2.65% to 3.99% and its coefficient of variation was 8.30%;The relative error of "bar" on TEM images of 15000 times was 3.82% ranging from3.43% to 4.54% and its coefficient of variation was 12.56%.The overall difference of the relative error was significant on 3500 times,8000 times and 15,000 times TEM image(F=58.862,P=0.000).
3.The morphometric analysis on the ultrastructural characteristics of A549 cells
(1)The observation and identificati on ultrastructure of A549 cell under TEM
A large number of short microvilli were presented on A549 cell surface. Transmission electron microscopy revealed various cellular components such as mitochondria, lysosomes,microvilli, as well as the nucleus
(2) The quantitative characteristics of A549 cell ultrastructure
①The quantitative characteristics of A549 nuclear and plasma
The VVn was 0.2789,its 95 per cent confidence interval ranged from 0.2451 to 0.3129 and its coefficient of variation was 0.2191.The VVcyt was 0.7211,its 95 per cent confidence interval ranged from 0.6871 to 0.7549 and its coefficient of variation was 0.0847.The Rnp was 63%.The SVn was 0.1934μm-1,its 95 per cent confidence interval ranged from 0.1785 to 0.2075μm-1 and its coefficient of variation was 0.2004. The vn was 465.76μm3,its 95 per cent confidence interval ranged from 409.25 to 522.27μm3 and its coefficient of variation was 0.2091.The vcyt was 1204.2μm3,its 95 per cent confidence interval ranged from 1147.7 to 1260.7μm3 and its coefficient of variation was 0.0847.The sn was 322.92μm2, its 95 per cent confidence interval ranged from 298.76 to 347.08μm2 and its coefficient of variation was 0.2004.
②The quantitative characteristics of A549 nucleoclus,euchromatin, hetero-chromatin
The VVnu was 0.0761, its 95 per cent confidence interval ranged from 0.0739 to 0.0781 and its coefficient of variation was 0.0571.The SVnu was 0.1508μm-1,its 95 per cent confidence interval ranged from 0.1424 to 0.1596μm-1 and its coefficient of variation was 0.1518.The vnu was 35.623μm3,its 95 per cent confidence interval ranged from 34.497 to 36.749μm3 and its coefficient of variation was 0.0571.The snu was 70.517μm2, its 95 per cent confidence interval ranged from 66.521 to 74.513μm2 and its coefficient of variation was 0.1518.The VVeuc was 0.7225,its 95 per cent confidence interval ranged from 0.7097 to 0.7363 and its coefficient of variation was 0.0372.The veuc was 337.85μm3,its 95 per cent confidence interval ranged from 331.73 to 343.97μm3 and its coefficient of va riation was 0.0372.The VVhet was 0.2012, its 95 per cent confidence interval ranged from 0.1883 to 0.2137 and its coefficient of variation was 0.1164. The vhet was 94.114μm3, its 95 per cent confidence interval ranged from 88.047 to 100.18μm3 and its coefficient of variation was 0.1164.
③The quantitative characteristics of A549 cell mitochondria
The Vvmi was 0.0460, its 95 per cent confidence interval ranged from 0.0443 to 0.0477 and its coefficient of variation was 0.0883.The SVmi was 0.5988μm-1,its 95 per cent confidence interval ranged from 0.5045 to 0.6935μm-1 and its coefficient of variation was 0.5638.The NVmi was 0.3003μm-3,its 95 per cent confidence interval ranged from 0.2296 to 0.3704μm-3 and its coefficient of variation was 0.4977.The vmi was 76.823μm3,its 95 per cent confidence interval ranged from 58.882 to 64.938μm3 and its coefficient of variation was 0.0883.The smi was 1001.67μm, its 95 per cent confidence interval ranged from 888.09 to 1115.3μm2 and its coefficient of variation was 0.5683.The nmi was 501,its 95 per cent confidence interval ranged from 384 to 618 and its coefficient of variation was 0.4977.
④The quantitative characteristics of A549 cell lysosomes, lamellar bodies and vesicles
The VVlam was 0.0250, its 95 per cent confidence interval ranged from 0.0162 to 0.0338 and its coefficient of variation was 0.6242.The VVlys was 0.1290, its 95 per cent confidence interval ranged from 0.0091 to 0.0119 and its coefficient of variation was 0.5177.The VVves was 0.0390, its 95 per cent confidence interval ranged from 0.0397 to 0.0401 and its coefficient of variation was 0.0466.The SVlam was 0.3565μm-1,its 95 per cent confidence interval ranged from 0.2715 to 0.4425μm-1 and its coefficient of variation was 0.6432.The SVlys was 0.1763μm-1,its 95 per cent confidence interval ranged from 0.0987 to 0.2533μm-1 and its coefficient of variation was1.1744. The SVves was 1.1612μm-1,its 95 per cent confidence interval ranged from 1.0485 to 1.2735μm-1 and its coefficient of variation was 0.2591.The NVlam was 0.5691μm-3,its 95 per cent confidence interval ranged from 0.4337 to 0.7043μm-3 and its coefficient of variation was 0.7127.The Nvlys was 0.1289μm-3, its 95 per cent confidence interval ranged from 0.0874 to 0.1706μm-3 and its coefficient of variation was 0.4624.The NVves was 10.424μm-3,its 95 per cent confidence interval ranged from 8.7206 to 12.274μm-3 and its coefficient of variation was 0.3178.The vlam was 41.763μm3,its 95 per cent confidence interval ranged from 62.384 to 91.257μm3 and its coefficient of variation was 0.6242. The vlys was 21.694μm3,its 95 per cent confidence interval ranged from 15.474 to 27.914μm3 and its coefficient of variation was 0.5177.The vves was 65.241μm3,its 95 per cent confidence interval ranged from 63.774 to 66.708μm3 and its coefficient of variation was 0.0406.The slam was 428.68μm2,its 95 per cent confidence interval ranged from 325.73 to 531.63μm2 and its coefficient of variation was 0.6432.The slys was 212.04μm2, its 95 per cent confidence interval ranged from 119.06 to 305.02μm2 and its coefficient of variation was 1.1744. The sves was 1396.2μm2, its 95 per cent confidence interval ranged from 1261.1 to 1531.3μm2 and its coefficient of variation was 0.2591.The nlam was 950, its 95 per cent confidence interval ranged from 724 to 1175 and its coefficient of variation was 0.7127.The nlys was 215,its 95 per cent confidence interval ranged from 146 to 284 and its coefficient of variation was 0.4624. The nves was 17,409, its 95 per cent confidence interval ranged from 14,564 to 20,253 and its coefficient of variation was 0.3178.
4. The comparative results of the morphological characteristics between nucleus and nucleolus of A549 and HBE cell s.
(1)The observation under cells seeding and HE staining and inverted microscopy.
A549 and HBE cells have visible difference in size and appearance. A549 cells was spindle-shaped and mainly scattered. HBE cells like polygonal epithelioid cells.
(2) The morphology of A549 and HBE cells under SEM and TEM.
Scanning electron microscope:The microvilla are presented on the surface of HBE cells and A549 cells. The irregular nuclei can be found under TEM observation of nuclei and nucleoli.Some nucleus contained two or more nucleolus. There are inclusions within both HBE cells and cells nucleus.
(3) The quantitative characteristics of A549 cells and HBE cells nucleus and nucleolus at three-dimensional level.
Based on the morphological parameters of HBE nucleus and nucleoli, the negative deviation of the volume density, nuclear surface area density, nuclear numerical density and the nuclear ratio surface to volume of A549 nuclear was 21.14%,27.96%,32.87% and 50% respectively. The positive deviation of the nuclear average volume, the average surface area of nuclear and the ratio of nuclear to cytoplasm of A549 were 146.98% and 59.65% and 12.90%.The negative deviation of A549 nucleolar volume density, surface area density, number density and the nucleolar ratio surface to volume was 7.81%,33.63%,36.64%and 43.68%.The positive deviation of A549 nucleolar average volume, the average surface area was 53.49% and 13.48% respectively.
Conclusion
1.The contraction level of A549 cells after treated with smearing and HE staining, smearing and Pap staining, paraffin-embedded and HE staining, transmission electron microscopy sample preparation and HE staining processing were large:66%,67%,69%, 71% respectively. The average diameter of A549 cell in different method are different.
2.There was the error between the length of "bar" on TEM image and the actual length of "bar".The error of "bar" was difference when magnification was different. The relative error of "bar" on the 3500 times,8000 times and 15,000 times images were 3.04%,2.77% and 3.82% respectively. The error of the "bar" on images of 15,000 times,8,000 times and 3,500 times was significantly difference.
3.The quantitative characteristics of A549 cell ultrastructure
(1)The individual A549 cell contained 72% cytoplasm and 28% nucleus.The nucleus contained about 8% nucleolus and about 92% chromatin,of which about 76.9% appear in the euchromatin form. About 4.6% of cytoplasm was made up of mitochondria. The lamellar body space amounted to 2.5% of cytoplasm, while autolysosomes and vesicles contributed 1.39% and 3.9% of cytoplasm respectively. Total mentioned organelles occupied about 15% of cytoplasm.
(2) In invidual A549 cell total surface area of mitochondria was 1001.67μm2 and the number of mitochondria was about 501.Total surface area of lamellar body was 428.68μm2 and the number of lamellar body was 950. Total surface area of lysosome was 212.04μm2 and the number of lysosome was approximately 215.Total surface area of vesicles was 1396.2μm2 and the number of vesicles was about 17409. 4. There was deviation between the morphology of A549 nucleus and nucleolus and HBE nucleus and nucleolus.The average degree of deviation of A549 nuclear was 56.43%.The average degree of deviation of A549 nucleolar was 31.46%.
New points
1.Through stereological studies on the ultrastructure of A549 cell we found the individual A549 cell contained 72% cytoplasm and 28% nucleus. The nucleus contained about 8% nucleolus and about 92% chromatin, of which about 76.9% appear in the euchromatin form.About 4.6% of cytoplasm was made up of mitochondria. The lamellar body space amounted to 2.5%of cytoplasm, while autolysosomes and vesicles contributed 1.39% and 3.9% of cytoplasm respectively. Total mentioned organelles occupied about 15% of cytoplasm.In invidual A549 cell total surface area of mitochondria was 1001.67μm2 and the number of mitochondria was about 501.Total surface area of lamellar body was 428.68μm2 and the number of lamellar body was 950.Total surface area of lysosome was 212.04μm2 and the number of lysosome was approximately 215.Total surface area of vesicles was 1396.2μm2 and the number of vesicles was about 17409.
2.The contraction level of A549 cells after treated with smearing and HE staining,smearing and Pap staining, paraffin-embedded and HE staining, transmission electron microscopy sample preparation and HE staining processing were large: 66%,67%,69%,71% respectively. The average diameter of A549 cell in different method are different.
3.There was the error between the length of "bar" on TEM image and the actual length of "bar".The error of "bar" was difference when magnification was different. The relative error of "bar" on the 3500 times,8000 times and 15,000 times images were 3.04%,2.77% and 3.82% respectively. The error of the "bar" on images of 15,000 times,8,000 times and 3,500 times was significantly difference.
4.There was deviation between the morphology of A549 nucleus and nucleolus and HBE nucleus and nucleolus.The average degree of deviation of A549 nuclear was 56.43%.The average degree of deviation of A549 nucleolar was 31.46%.
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