| Background and objectiveA549 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. Methods1.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.Results1.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 TEMA 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 ultrastructure①The quantitative characteristics of A549 nuclear and plasmaThe 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-chromatinThe 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 mitochondriaThe 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 vesiclesThe 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.Conclusion1.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 points1.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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