Çѹø ¼¿¶óÀÎ(cell line)ÀÌ È®¸³(estalblished)µÇ¸é, ±×°ÍÀº Ư¼ºÈ(characterised)µÈ´Ù.
±×°ÍÀº Á¾(species)ÀÇ È®¸³(confirm)°ú Á¶Á÷(tissue)ÀÇ º»¼º(origin)¶§¹®ÀÌ´Ù.
--> fibroblast°¡ ¹èÁö(culture)¿¡¼ Àß ÀÚ¶ó ´Ù¸¥ ¼¼Æ÷ Á¾·ù(cell type)À» ¿À¿°È(contaminate)ÇÏ´Â ¹®Á¦Ã³·³ »óÈ£ ¿À¿°È¿©ºÎ(no cross-contamination)ÀÇ È®ÀÎ(check)ÀÌ ÇÊ¿äÇÏ´Ù.
* ¼¿¶óÀÎ(cell line)Àº ÁÖ±âÀûÀ¸·Î »óÈ£ ¿À¿°È ¿©ºÎ(no cross-contamination)°ú ºÒ¾ÈÁ¤¼º(instability), ´Ù¸¥ ¼¿Æ¯¼ºÀ¸·Î ºÐÈÇÏ´Â ¼ºÁú(variation)¿¡ ´ëÇØ È®ÀÎ(check) ÇØ¾ß ÇÑ´Ù.
4.2. Á¾ÀÇ ´Ù¾ç¼º(Species varification)
4.2.1 Cytogenetics : heredity(À¯Àü¼º)¿Í variation(ºÐȼº)°úÀÇ chromosomal structure(¿°»öü ±¸Á¶)ÀÇ correlation(»óÈ£¿¬°ü¼º).
* speciesÀÇ most characteristic trait(Ư¡Àû Ư¼º)´Â ±×°ÍÀÇ chromsome content(¿°»öü ±¸¼º¿ä¼Ò)ÀÌ´Ù.
* chromosomal analysis(¿°»öü ºÐ¼®)´Â species(Á¾) »çÀÌÀÇ most reliable distinguish method(°¡Àå ½Å·ÚÇÒ ¸¸ÇÑ ±¸ºÐÀû ¹æ¹ý)ÀÌ´Ù.
* karyotype : chromosomal number(¿°»öü ¹øÈ£), size(Å©±â), shape(¸ð¾ç)¿¡ ¾²ÀÌ´Â term(¿ë¾î)
# karyotype techniques : chromosome(¿°»öü)ÀÇ size¿Í shape´Â nuclear division stage(ÇÙ ºÐ¿ ´Ü°è)¿¡¼ ´Ù¾çÇÏ´Ù.
a. cell in mid-lag phase
b. incubated(¹è¾ç) with colcemid or vinblastine (meta phase¿¡ cellÀ» arrest(ÀâÀ½))
c. cell division(ºÐ¿) inhibited(¾ïÁ¦) at metaphase
d. trypsinise (anchorage-dependent animal cell (ºÎÂø¼º µ¿¹° ¼¼Æ÷)À» ¹è¾ç¿ë±â ¹Ù´Ú¿¡¼ ¶¼¾î³¿)
e. cell suspension
f. incubate in hypotonic(KCl) solution (ÀúÀå¾×¿¡¼ ¹è¾ç)
g. swollen cell (¼¼Æ÷¸¦ ÅͶ߸²)
h. incubate with methanol : acetic acid (3:1 v/v) at 4¢ªC to fix cells
i. fixed cell (¼¼Æ÷¸¦ °íÁ¤)
j. centrifuge (¿ø½ÉºÐ¸®)
k. cells collected and fropped onto a slide (¼¼Æ÷¸¦ ¸ðÀ¸°í ½½¶óÀ̵忡 ¶³¾î¶ß¸²)
l. dry (air or heat dried)
m. cells attached to slide (½½¶óÀ̵忡 ¼¼Æ÷¸¦ ºÎÂø½ÃÅ´)
n. immerse in stain under appropriate conditions (Àû´çÇÑ È¯°æ¿¡¼ ¿°»ö)
o. slide viewed and photographed (½½¶óÀ̵忡 ³õ°í »çÁøÃÔ¿µ)
# Giemsa dye (=G-banding) pH 6.8¿¡¼ staining(¿°»ö)
chromosome(¿°»öü) stain(¿°»ö)µÇ¾î bright³ª dull fluorescent band(È帴ÇÑ Çü±¤¶ì)³ªÅ¸³»¾î chromosome(¿°»öü)ºÐ¼®¿¡ ¾²ÀÌ´Â reagentÀÌ´Ù.
# Q-banding (Quinacrine banding)
interband regionÀ» stainÇÏ´Â quinacrine mustard³ª quinacrine dihydrochloride¸¦ »ç¿ëÇÏ´Â ¹æ¹ý
human(Àΰ£)¿¡¼ Y chromosome(Y ¿°»öü)ÇÏ°Ô Çü±¤ÇÏ¿© G-banding¿¡¼ Y chromosomeÀ» dull uniformÇÏ´Â °Í°ú ´Ù¸£¹Ç·Î particular value(Ưº°ÇÑ Áö½ÃÁ¡)°¡ ÀÖ´Ù.
# C-banding
centromore regionÀ» stainÇÏ¿© G-banding¿¡¼ ºÒºÐ¸íÇÑ Áö¿ªÀ» clarify(È®½ÇÇÏ°Ô) ÇÑ´Ù.
# G11-banding
pH 11¿¡¼ Giemsa staning ÇÏ´Â ¹æ¹ý
* mouse chromosomeÀÇ pale blue centromere¸¦ dark margenta(½ÉÈ«»ö)·Î stain(¿°»ö) ÇÑ´Ù.
* human chromosomeÀÇ chromosome number(1,4,5,7,10,13,14,15,20,21,22)ÀÇ red region¿¡ blue·Î stain ÇÑ´Ù.
--> mouse/human hybrid, Áï, hybridomas¸¦ ÃøÁ¤ÇÏ´Â value(ÃøÁ¤Ä¡)¸¦ Á¦°øÇÑ´Ù.
4.2.2. Isoenzymology
animal tissue(µ¿¹°Á¶Á÷)¿¡¼ ¾î¶² enzymeÀº multiple form(´Ù¾çÇÑ ±¸Á¶)À¸·Î Á¸ÀçÇÑ´Ù.
# Isoenzyme(=isozyme)
°°Àº reaction(¹ÝÀÀ)À» catalysis(Ã˸Å)ÇÏ´Â enzymeÀÇ different molecular form(´Ù¸¥ ºÐÀÚÀû ±¸Á¶)
chromatography³ª electrophoresis(Àü±â¿µµ¿)¸¦ ÅëÇØ isoenzymeÀ» seperate(ºÐ¸®) ÇÒ ¼ö ÀÖ´Ù.
--> enzyme bandÀÇ ¼ö³ª intensity´Â speciesÀÇ indicativeÀÌ°í cell lineÀÇ lineage ÀÌ´Ù.
È®¸³ÇÑ band¸¦ ´Ù¸¥ speciesÀÇ conventional band patternÀ» º¸¿©ÁÖ´Â publication(ÀÌ¹Ì ³ª¿ÍÀÖ´Â ÀÚ·á)°ú ºñ±³ÇÑ´Ù.
ex) Esterase enzymeÀÇ ºñ±³
{human kidney},{HeLa,human skin, Human heart}, {L5178Y(mouse)}ÀÇ gel bandÀÇ ºñ±³
agarose cassette system¿¡ ÀÇÇÑ ´Ù¸¥ isoenzymeÀÇ mobilities(¿òÁ÷ÀÓ)ÀÇ determination(°áÁ¤)Àº cell lineÀÇ identity(±¸ºÐÁ¡)¸¦ establish(°áÁ¤Áþ´Â)ÇÏ´Â È¿°úÀû ¹æ¹ýÀÌ´Ù.
--> ºñ±³Àû Á¤È®ÇÏ°í ºü¸£¸ç less expensive(°ªÀÌ ºñ½ÎÁö ¾Ê´Ù)ÇÏ´Ù.
# glucose 6-phophatate dehydrogenase(G6PDH)
* AÇü - caucasian(¹é»öÀÎÁ¾), 80% negroids(ÈæÀÎ)
* BÇü - 20% negroids, HeLa(ÀڱþϿ¡¼ È®¸³µÈ ¼¼Æ÷ÁÖ)
# Lactose dehydrogenase(LDH)
# nucleoside phophorylase(NP)
# esterase
4.2.3. Immunological tests (¸é¿ªÇÐÀû Å×½ºÆ®)
immunofluorescence(¸é¿ªÇü±¤)¿¡ ÀÇÇØ species-specific antigen(Á¾ ƯÀÌÀû Ç׿ø)Àº detect(°ËÃâ) µÉ ¼ö ÀÖ´Ù.
specis specific binding(Á¾ ƯÀÌÀû °áÇÕ)ÇÏ´Â antisera(Ç×Ç÷û)¿¡ fluorescein isothiocyanate°¡ ºÙ¾îÀÖ´Ù.
ÀÌ°ÍÀ» cell membrane(¼¼Æ÷¸·)°ú mixed(¼¯´Ù) ÇÏ¿© incubate(¹è¾ç) ÇÑ ´ÙÀ½ washed and mounted on slide Çϸé antisera(Ç×Ç÷û)°¡ membrane(¸·)ÀÇ ¹ÝÀÀÇÏ´Â recepter(°áÇÕÀÚ)¿¡ binding(°áÇÕ)ÇÏ°Ô µÈ´Ù.
UV fluorescence microscope(¹æ»ç¼± Çü±¤ Çö¹Ì°æ)¸¦ Çϸé positive reaction(¾ç¼º ¹ÝÀÀ)ÀÌ µÇ¸é cell membrane(¼¼Æ÷ ¸·)¿¡ bright green peripheral fluorescence(¹àÀº ³ì»ö Çü±¤)°¡ ³ªÅ¸³²
4.3. Intra-species contamination (³»ºÎ Á¾ ¿À¿°)
cell lineÀÇ specis originÀÌ confirm(È®¸³)µÇ¸é, specis¿¡¼ derived(À¯·¡) µÈ cell line »çÀÌ¿¡¼ individual(°³Ã¼)À» ¹àÈ÷´Â°Ô Áß¿äÇÏ´Ù.
individual genetic information(°³Ã¼ À¯Àü Á¤º¸)ÀÇ most circumstance(ȯ°æ)´Â unique(À¯ÀÏ)ÇÏ´Ù
--> Á÷Á¢ÀûÀÎ genetic material(À¯ÀüÀû ¹°Áú)ÀÇ examination(°Ë»ç), genetic materialÀÇ protein product(´Ü¹éÁú »ý»ê¹°)ÀÇ investigation(Ž»ö)À¸·Î ¹àÈû
4.3.1. Cytogenetics ; genome analysis (À¯Àü ºÐ¼®)
banding patternÀº ¾î´À speciesÀÇ different member¿¡¼ characteristicÇÏ´Ù.
--> individual(°³Ã¼) »çÀÌÀÇ difference´Â karyotypeÀÇ ºñ±³¿¡ ÀÇÇØ recognised(È®ÀÎ)°¡ °¡´ÉÇÏ´Ù. crudeÇÑ ¹æ¹ý
4.3.2. Recombinant DNA methods ; genome analysis (ÀçÁ¶ÇÕ DNA ¹æ¹ý)
gene(À¯ÀüÀÚ) ±× ÀÚü¸¦ typing(ºÐ¼®) ÇÔÀ¸·Î¼ individual's genotype(°³Ã¼ÀÇ À¯ÀüÀÚ Å¸ÀÔ)À» Á÷Á¢ determine(°áÁ¤) ÇÒ ¼ö ÀÖ´Ù.
# polymorphism
¿©·¯ gene¿¡¼ specisÀÇ individual(°³Ã¼) »çÀÌ¿¡ slight variation(¾à°£ÀÇ Â÷ÀÌÁ¡)ÀÌ ÀÖ´Ù.
particular gene(Ưº°ÇÑ À¯ÀüÀÚ)ÀÇ DNA¿¡¼ molecularÀÇ difference(Â÷ÀÌ)¸¦ ¸»ÇÑ´Ù.
--> DNA bases(¿°±â)ÀÇ dissimilar arrangement(´Ù¸¥ ¹è¿)ÀÇ Á¸Àç´Â °¢°¢ÀÇ individual's DNA(°³Ã¼ÀÇ DNA)°¡ °°Àº restriction enzyme(Á¦ÇÑÈ¿¼Ò)¿¡ ÀÇÇØ ´Ù¸¥ distinct length(Â÷ÀÌ°¡ ³ª´Â ±æÀÌ)ÀÇ restriction fragment(ºÐÇØµÈ Á¶°¢)ÀÌ ³ª¿À´Âµ¥, ÀÌ´Â slight different site(¾à°£ ´Ù¸¥ ºÎºÐ)¿¡¼ À߸°´Ù´Â °ÍÀ» ÀǹÌÇÑ´Ù.
# RFLP ( restriction fragment length polymorphism)
ÀÌ polymorphismÀ» detect(ã±â)Çϱâ À§ÇØ DNA probe(ŽħÀÚ)°¡ »ç¿ëµÉ ¼ö ÀÖ´Ù.
°°Àº Á¦ÇÑÈ¿¼Ò·Î ó¸®Çصµ polymorphism¿¡ ÀÇÇØ »ý±â´Â DNA fragment(Á¶°¢)¿¡¼ ´Ù¸¥ º¯ÀÌ°¡ »ý±è.
# Finger printing
a. cellular DNA
b. digestion with restriction enzyme (ºÐÇØÈ¿¼Ò·Î ºÐÇØ)
c. DNA fragment seperated (DNA Á¶°¢ ºÐ¸®)
d. southern blotting
e. DNA fragments transferred(¿Å±è) to membrane (membraneÀÇ DNA´Â meltedeµÈ Áï, ¹ÝÀÀÀÌ °¡´ÉÇÑ »óÅÂÀÎ single strandÀÌ´Ù.)
f. DNA melted(³ìÀÓ) and incubated(¹è¾ç) with radioactive probes(¹æ»ç¼± ŽħÀÚ)
g. probe hybridise(ŽħÀÚ È¥¼ºÈ) with DNA fragments carrying complementary(»óº¸Àû) sequence
h. wash and place next to photographic film
i. autoradiiogram developed
4.3.3. High resolution 2-D electrophoresis
distinctive gene(ƯÀÌ À¯ÀüÀÚ)ÀÇ consequence´Â °¢°¢ individualÀÇ proteinÀÇ characteristic patternÀ¸·Î ³ªÅ¸³´Ù.
--> cell line¿¡ ÀÇÇÑ protein, Áï, Àß ¾Ë·ÁÁø specific proteinÀÇ probe¿¡ ÀÇÇÑ °Í°ú ¸ðµç proteinÀÇ analysis¸¦ ÅëÇØ Â÷ÀÌÁ¡À» ¹ß°ßÇÒ ¼ö ÀÖ´Ù.
a. IEF(isoelectric forcusing) - 1¢ª dimension
urea Á¸ÀçÇÏ¿¡ molecular weight(ºÐÀÚ·®)°ú isoelectric point¸¦ ÀÌ¿ëÇÏ¿© cellÀÇ different proteinÀ» ÀÏÂ÷¿øÀ¸·Î seperate(ºÐ¸®) ÇÑ´Ù.
b. SDS-page (sodium dodecyl sulfate polyacrylamide gelelectrophoresis) - 2¢ª dimension
detergent sodium dodecyl sulfate(SDS) Á¸Àç ÇÏ¿¡ electrophoresis(Àü±â¿µµ¿)¸¦ ÇÑ´Ù.
protein size¸¦ ±âÃÊ·Î ºÐ¸®ÇÔ.
--> single sample¿¡¼ 10,000 proteinÀ» resolve(ºÐ¼®) ÇÒ ¼ö ÀÖ´Ù.
°¢ cell line¿¡ ¸Å¿ì specificÇÑ fingerprint¸¦ Á¦°øÇÑ´Ù.
4.3.4. Allozyme analysis - allelic isoenzyme ºÐ¼®
°°Àº specisÀÇ ´Ù¸¥ individualÀº ÁÖ¾îÁø enzyme locus(À§Ä¡)¿¡¼ ´Ù¸¥ allele¸¦ Ç¥ÇöÇÑ´Ù.
--> allozymeÀÇ ºÐ¼®Àº cell lineÀÇ genetic signature¿Í °°´Ù.
* allele : chromosomeÀÇ °°Àº locus¿¡¼ ¹ß°ßµÇ´Â geneÀÇ ´Ù¾çÇÑ formÀ̳ª ´Ù¸¥ form
4.3.5. Blood group antigens and HL-A (human lymphocyte antigen)
* cellÀÇ surface¿¡ Á¸ÀçÇÏ´Â antigenÀ» determination(°áÁ¤) ÇÑ´Ù.
* Blood group antigenÀº primary culture¿¡¼ÀÇ normal human epithelium¿¡¼¿Í ¾î¶² continuous epithelial line¿¡ Á¸ÀçÇÑ´Ù.
--> antiserumÀ» typeÇÏ´Â anti-human A, B, AB ¸¦ »ç¿ëÇÏ¿© cell lineÀÇ typeÀÌ °¡´ÉÇÏ´Ù.
* major histocompatibility antigen À̳ª major histocompatibility complex(HMC)
: °¢°¢ÀÇ »ç¶÷¿¡¼ immunological individuality(¸é¿ªÇÐÀû °³Ã¼¼º)¿¡ ¹ÝÀÀÇÏ´Â ¸Å¿ì ´Ù¾çÇÑ antigen nucleated cellÀÇ plasma membrane¿¡ Á¸ÀçÇÑ´Ù.
--> ÇÑ individual¿¡¼ °ü·Ã¾ø´Â individual¿¡ transplanted(Àü´Þ)µÈ tissue´Â foreignÀ¸·Î ÀνÄÇÏ¿© recipient's immune system¿¡ ÀÇÇØ Á¦°ÅµÈ´Ù.
* Human Lymphocyte Antigens(HL-A) system (=histocompatibility antigen)
chromosome(¿°»öü)¿¡ HL-A antigen(Ç×ü)À» coding(¾ÏÈ£È)ÇÏ´Â gene(À¯ÀüÀÚ)ÀÌ ÀÖ´Ù.
¾î¶² locus¿¡¼ geneÀº Ç×»ó °°Áö¸¸Àº ¾Ê´Ù. °¢ geneÀÇ ´Ù¸¥ formÀº alleleÀÌ´Ù.
two stage complement-dependent cytotoxicity test¸¦ ÀÌ¿ëÇÑ ÀÌ alleleÀÇ typingÀº cell lineÀÇ identificationÀ¸·Î specificationÀ¸·Î specific systemÀ» Á¦°øÇÑ´Ù.
4.4. Characterisation of cell type and stage of differentiation
4.4.1. The organization of cells in vivo
* in vivo¿¡¼ cellÀº tissue(ºñ½ÁÇÏ°Ô specific functionÀ» ¼öÇàÇϰųª function groupÀ» Çü¼ºÇÑ specialised cellÀÇ group, extracellular matrix¿Í associatedµÈ)·Î Á¶Á÷ȵȴÙ. °¢ tissueÀÇ Á¾·ù´Â characteristic size, shape, arrangementÀÇ cell·Î ±¸¼ºµÈ´Ù.
* ¿©·¯ typeÀÇ tissue°¡ organÀ» Çü¼ºÇÏ°í organÀº organ systemÀ¸·Î arranged µÈ´Ù.
* complex animalÀº lymphatic system, the respiratory system, the digestive system, the endocrine system °°ÀÌ ¸Å¿ì ´Ù¾çÇÑ organ systemÀ» °¡Áø´Ù.
* most cellÀÇ surface ȯ°æÀº °¢ tissue¿¡ specificÇÑ glycoprotein°ú proteoglycanÀÇ complex mixtureÀÌ´Ù.
1. Connective tissue (=stroma, stroma cell)
* bodyÀÇ tissue¿¡ ÇÔ²² joinµÇ¸é, body¸¦ support, underlying organÀ» protect ÇÑ´Ù.
* extracellular matrix°¡ »ó´ëÀûÀ¸·Î ÀûÀº cellÀÌ Æ÷ÇÔµÇ¾î ±¸¼ºÇÑ´Ù.
* adipose tissue, cartilage and bone, fibroblast(extracellular matrix component¸¦ Á¦°øÇÏ´Â cell)
2. Epithelial tissue
* body surface¸¦ coveringÇϰųª body »çÀÌÀÇ cavity¸¦ liningÇÏ´Â cellÀÇ sheet³ª continuous layer¸¦ Çü¼ºÇϱâ À§ÇØ ÇÔ²² tightly fitted cell·Î ±¸¼ºµÈ´Ù.
* ÇÑ sheetÀÇ surface´Â epithelial cell¿¡¼ »ý»êÇÏ´Â polysaccharide materialÀÇ tiny fibres·Î ±¸¼ºµÈ non-cellular basement membrane¿¡ ÀÇÇØ underlying tissue¿¡ ºÎÂøµÈ´Ù.
* ¸ð¾ç
- squamous(ºñ´Ã¸ð¾ç) : ÀÔÀ̳ª blood vesselÀÇ lining
- cuboidal(ÀÔ¹æÇü) : gland¿Í duct
- columnar(´Ù°¢ÁÖÇü) : small intestineÀÇ lining
- single layer(simple), two or more layer(stratified)
* ¸¹Àº epithelial cellÀº milk, mucus, sweat °°Àº specific product¸¦ ºÐºñÇϴµ¥ speciallised µÇ¾î ÀÖ´Ù.
3. Muscular tissue
* contraction¿¡ specialisedµÈ cell·Î ±¸¼º
* actiinÀ̳ª myosinÀ¸·Î ±¸¼ºµÈ myofibril·Î ºÒ¸®´Â contractile fibres¸¦ Æ÷ÇÔ
* cellÀÇ location°ú property¿¡ µû¶ó skeletal, smooth or cardiacÀ¸·Î ºÐ·ù
smooth (internal organ, bloodvessels, duct), striated(skeleton and associated)
4. Nervous tissue
* conducting impulse³ª cellÀ» connectiongÇϰųª supportingÇϴµ¥ associatedÇϴµ¥ specialisedµÈ cell·Î ±¸¼º
* brain, spinal cord, peripheral nerve, ganglia cellÀÌ Æ÷ÇÔÇÏ°í ÀÖ´Ù.
* neuron : nervous systemÀÇ actual conducting cell, cell body, dendrites, axonÀ¸·Î ±¸¼º
* glial cell : connecting and supporting cell
ex. Schwan cell : peripheral nerve¸¦ envelopeÇÏ´Â cell, nerve axonÀ» insulateÇÏ´Â myelin sheath¸¦ »ý»êÇÑ´Ù.
5. blood and lymph tissue
peripheral blood, bone marrow¿Í lymph gland¿¡¼ precusor cell
ex) °¡½º±³È¯(O©ü¿Í CO©ü), immune response
erythrocyte, white blood cell (granulocyte, monocyte, lymphocyte, natural killer cell), pletelet
4.4.2. Cell division and differentiation in vivo
totipotent ovum (¹«¾ùÀÌµç µÉ ¼ö ÀÖ´Â »óÅÂÀÇ cell) --> cell division and differentiation --> pluripotent stem cell (¸î°¡Áö ¼¼Æ÷·Î ºÐÈ°¡ °¡´ÉÇÑ »óÅÂÀÇ cell) --> cell division and differentiation --> unipotent cell (ÇÑ°¡Áö cell typeÀ¸·Î¸¸ ºÐÈ°¡ °¡´ÉÇÑ »óÅÂÀÇ cell) --> cell division and differentiation --> mature differentiated cell
* young animal¿¡¼± cell multiplicationÀÌ cell death¸¦ ÃÊ°úÇϹǷΠanimal cellÀÇ ¼ö°¡ Áõ°¡ÇÑ´Ù.
* adult¿¡¼± cell division°ú cell death°¡ steady state·Î balance ÇÑ´Ù.
* cellÀº mitosis¿Í cell cycle¿¡ °ü·ÃµÈ cytokinesis¸¦ ÅëÇØ multiply ÇÑ´Ù.
haematopoietic system¿¡¼ pluripotent undifferentiated stem cellÀÇ small populationÀº terminally differentiated cell·Î ÁøÇàµÇ´Â commited precursor cell·Î ÀÏÀ¸Å³ ¼ö ÀÖ´Ù.
terminally differentiated cell(phenotype fully expressed, ´õ ÀÌ»ó preceed ÇÒ ¼ö ¾ø´Â cell)·Î ÁøÇàµÇ´Â commintted precusor cell·Î ÀÏÀ¸Å³ ¼ö ÀÖ´Ù.
stem cell : divide without limit and give rise to differentiated cells
4.4.3. Factors needed for differentiation in vivo
´Ù¸¥ typeÀÇ cell »çÀÌÀÇ interactionÀº normal development µ¿¾È differentiationÀ» promoteÇÏ´Â °Í°ú initiatingÇÏ´Â °ÍÀ» À¯¹ßÇÑ´Ù.
ex) epithelial precusor¿Í stromal precusor °£ÀÇ interactionÀº epithelial differentiationÀ» induce ÇÑ´Ù.
--> cell-cell interactionÀº adult organism¿¡¼ differentiation µ¿¾È roleÀ» ¼öÇà
a. hormone
endocrine glands¿¡¼ bloodstreamÀ¸·Î Á÷Á¢ÀûÀ¸·Î ºÐºñµÇ´Â intercellular signalling molecule Áï, substance¸¦ ¸»ÇÔ --> ´Ù¸¥ cell¿¡ ¿µÇâÀ» ³¢Ä§.
* endocrine signalling : ¸Ö¸® ÀÖ´Â cell¿¡µµ ¿µÇâÀ» ³¢Ä§
b. paracrine factors (paracrine signalling :°¡±îÀÌ ÀÖ´Â ÀÎÁ¢ÇÑ ¼¼Æ÷¿¡ ¿µÇâÀ» ³¢Ä§)
* bloodstreamÀ» ÅëÇØ À̵¿ÇÏÁö ¾Ê´Â´Ù.
* signalling cell¿¡ °¡±î¿î target cell¿¡ ¿µÇâÀ» ÁØ´Ù.
* in vivo¿¡¼ development³ª differentiation¿¡¼ ±â´É ¼öÇà
c. extracellular matrix
* collagen°ú non-collagenous protein(laminin, fibronectin) ±×¸®°í carbohydrate-rich moleculeÀÇ mixtureÀÌ´Ù.
* cellular matrixÀÇ modification¿¡ ÀÇÇØ cell differentiationÀÌ À¯µµµÈ´Ù.
ex) extracellular matrix proteinÀÎ tenascinÀº stromal cell¿¡¼ ÇÕ¼ºµÇ´Âµ¥ in vivo¿¡¼ epithelial tissueÀÇ differentiationÀ» À¯µµÇÑ´Ù.
* normal growth substratumÀ» Çü¼ºÇÏ°í differentiationÀÇ À¯µµ¿¡¼ essential role ¼öÇà.
d. cell-cell contact
* gap junction (intracellular communication¿¡ Áß¿ä)
°³Æó°¡ °¡´ÉÇÑ connexonÀ̶ó ºÒ¸®´Â hexagonal protein subunit¿¡ ÀÇÇØ Çü¼ºµÈ large channel.
--> most eukaryote membrane¿¡ Á¸ÀçÇϸç, ÀÌ¿ôÇÑ cellÀÇ interior »çÀÌÀÇ passage way·Î serveÇÑ´Ù.(cell »çÀÌÀÇ ionÀ̳ª small moleculeÀÇ È帧À» Çã¿ë)
¡Å development³ª differentiation¿¡ ÇʼöÀû
* cellÀº surface informationÀÇ exchange¸¦ ÅëÇØ interactÇÑ´Ù.
ÇÑ cellÀÇ surface¿¡ Ç¥ÇöµÈ moleculeÀº Á÷Á¢ÀûÀ¸·Î adjacent cellÀÇ behavior¿¡ ¿µÇâÀ» ³¢Ä£´Ù.
connection pore¸¦ ÅëÇØ ºÐÈ¿¡ °ü°èµÈ ¹°ÁúÀ» ÁÖ°í ¹ÞÀ½
1000 ~ 1500 daltonº¸´Ù ÀÛÀº ºÐÀÚµéÀÌ connexon poreÀ» ÅëÇØ À̵¿ÇÏ¿© communication ÇÔ.
ec) sugar, amino acid, vitamin, nucleotids
4.4.4. Animal cell types grown in vitro
* fibroblast (¼¶À¯¾Æ¼¼Æ÷)
* epithelial tissue (Ç¥ÇÇÁ¶Á÷) ; liver, lung, breast, skin, bladder, kidney cell
* neural cell (±³ÁÖ¼¼Æ÷) ; glial cell, neuron(in vitro¿¡¼ proliferate ÇÏÁö ¸øÇÔ)
* skeletal cell, cardiac cell, smooth muscle cell
* skeletal tissue ; bone cell, cartilage cell
* endocrine cell ; adrenal cell, pituitary cell, pancreatic islet cell
* melanocytes
* tumor cell
4.4.5. Differentiated state in vitro
in vitro¿¡¼ ÀÚ¶ó´Â cellÀÌ ¿©ÀüÈ÷ culture¿¡¼ ±×µéÀÇ differntiated characteristicÀ» °¡Áö°í ÀÖ´ÂÁö confirmÇÏ´Â°Ô Áß¿äÇÏ´Ù.
a. connective tissue
cultured fibroblast´Â ÇüÅÂÀûÀ¸·Î fibroblast tissueÀ̳ª differentiatied°¡ ´ú µÇ·Á´Â °æÇâÀÌ ÀÖ°í fibroblast precursor°¡ ³ªÅ¸³´Ù.
cultures fibroblast°¡ ºÐÈÇϸé adipose tissue¿Í connective tissue·Î µÈ´Ù.
b. epithelial tissue
# fibroblastó·³ plastic dish¿¡ tightÇÏ°Ô ºÙ¾î ÀÚ¶õ´Ù. ±×¸®°í, laminin °°Àº extracellular matrix proteinµµ ºÐºñÇÑ´Ù.
# ÇÏÁö¸¸, culture¿¡¼ Áß¿äÇÑ characteristic¸¦ ÀҴ´Ù.
* differentiated productÀÇ expression levelÀº hormone controlÀ» ¹Þ´Â´Ù.
--> fullly differentiated epithelial cellÀº culture¿¡¼ 3ÀÏ »çÀÌ¿¡ tissue-specific functionÀ» ÀҴ´Ù.
# De-differentiated cellÀº chracteristic substrate³ª ´Ù¸¥ cell typeÀÇ Á¸ÀçÇÏ´Â ¿©°Ç¿¡¼ ´Ù½Ã normal state·Î returnÇÒ ¼ö ÀÖ´Ù.
ex) mammary epithelial cellÀº fat pad³ª collagen type I gel¿¡¼ ÀÚ¶ö¶§ normal ductal morphogenesis¸¦ º¸ÀÌ°í Àû´çÇÑ hormoneÀ» ÷°¡½Ã milk proteinÀ» ºÐºñÇÑ´Ù.
--> cell-cell°ú cell-extracellular matrix interactionÀÌ differentiationÀ» À¯µµÇÏ´Â soluble factoró·³ Áß¿äÇÔ
ex) skin epithelial cellÀº epithelial growth factor¿Í ´Ù¸¥ cell typeÀÇ feeder layer(growthÁõÁø, differentiation ÁõÁø)¿¡¼ ºÐÈÀ¯ÁöÇÏ¸ç ¼ºÀåÇÑ´Ù.
* feeder layer : paracrine factor¿Í extracellular matrix, ´Ù¸¥ cell typeÀÇ differentiation¿¡ ÇÊ¿äÇÑ Àû´çÇÑ interactionÀ» Á¦°øÇÏ´Â cell surface molecule¸¦ Á¦°øÇÑ´Ù.
c. Muscular tissue
# mycoblast
* permament lines of muscle precusor cell
* developÇÏ¿© certain density¿¡ µµ´ÞÇϸé, in vivo¿¡¼¿Í ºñ½ÁÇÑ muscle cell functionÀ» ¼öÇà --> ÀÏÁ¤ÇÑ ¼¼Æ÷ ³óµµ¿¡¼ in-vivo¿Í À¯»çÇÑ ±â´É º¸ÀÓ
* ¼·Î fuseÇÏ¿© cross-striated multi-nucleated muscle fibres(myotubes) Çü¼º
d. Nervous tissue
* in vitro¿¡¼ neural cellÀº ±×µéÀÇ typical neural morphology¸¦ À¯ÁöÇÑ´Ù.
* nervous systemÀ» ±¸¼ºÇÏ´Â glial cell°ú neuralÀÇ heterogeneity´Â ±×µéÀÇ individual subclassesÀÇ physiology¿Í biochemistry¸¦ ÀÌÇØÇϴµ¥ À庮ÀÌ ÀÖ´Ù.
e. Blood and lymph
leukocytes(lymphocytes, monocytes, macrophage)ÀÇ precursor´Â culture¿¡¼ ÃÖ¼ÒÇÑÀÇ differentiated functionÀ» ¼öÇàÇÑ´Ù.
ex) red blood cellÀÇ abnormal precusorÀÎ Erythroleukemia cellÀº haemoglobin »ý»êÀ» À¯µµÇÏ°í normal red cell maturation°ú ¿¬°üµÈ cell membraneÀÇ structural change¸¦ ¼öÇàÇÑ´Ù.
4.4.6. Characterisation of cell types in vitro
a. Morphology (ÇüÅÂ)
# microscope¿¡¼ morphology °üÂû
* Epithelial cell : regular, polygonal, with clearly defined edge --> ´Ù°¢Çü
* Fibroblaste : bipolar, characteristic parallel array, whorls visible --> ¾ç±ØÈ
# morphology´Â »ç¿ëÇÑ medium°ú cultureµÃ cellÀÇ substrate¿¡ º¯ÈÇϹǷΠ°°Àº cell density, °°Àº medium, °°Àº substrate °°ÀÌ ¶È°°Àº growth stage¸¦ ¸¸µé¾î¾ß ÇÑ´Ù.
# ¼¼Æ÷ÀÇ originÀÌ ¾Æ´Ï¶ó apperance·Î ±¸ºÐÇÑ´Ù.
* fibroblast - like = fibroblastiod
* epithelial - like = epitheleoid
b. Intermediate filament proteins
# eukaryotic cellÀº cellular morphology¸¦ À¯ÁöÇÏ°í intracellular transportÀÇ ±â´ÉÀ» ÇÏ°í, cell motility, mitocis, meiosis, cytoskeletonÀÇ ÇʼöºÎºÐÀ» ±¸¼ºÇϹǷΠcell typeÀÇ identificationÀ̶ó ºÒ¸®´Â protein networdÀ» Æ÷ÇÔÇÑ´Ù.
--> cytoplasmic intermediate filament proteinÀº cytoskeletonÀÇ ÇʼöºÎºÐÀ» ±¸¼ºÇϹǷΠcell typeÀÇ identification¿¡ »ç¿ëÇÑ´Ù.
ex) cytokeratin --> epithelial cell
vimentin --> stromal cell
fibronectin --> fibroblast
desmin --> myogenic cell
neurofilament --> neuron, glial cell
¡Å cell typeÀ» ±¸ºÐÇϱâ À§ÇØ ´Ù¾çÇÑ intermediate filament protein¿¡ antibody¸¦ »ç¿ëÇÏ¿© immunoflurescene·Î cellÀ» Á÷Á¢ stain.
c. Cell surface antigen
lymphocytesÀÇ subspeciesÀÇ sorting¿¡ Ưº°È÷ À¯¿ëÇÏ´Ù. <-- monoclonal antibodyÀÇ availability¿¡ ÀÇÇؼ
d. Expression of functional properies of the mature cells.
in vivo¿¡¼ specialised functionÀº specific protein productÀÇ activity¸¦ Ç¥ÇöÇÑ´Ù.
ex) erythrocytes --> haemoglobin ³ª¿È
mammary epithelial cell --> milk protein ³ª¿È
e. Analysis of isoenzymes
# ¾î¶² cell lineÀº ¾î´À cell type specific enzymeÀ» ¹ßÇöÇÏÁö ¾Ê´Â´Ù. ±×·¯³ª, ¾î¶² housekeeping enzymeÀÇ isoenzymeÀÇ patternÀº cell-type¸¶´Ù chatacteristic ÇÏ´Ù.
ex) phosphoglucomutase(PGM1, 3), 6-phosphogluconate dehydrogenase(PGD), malic enzyme(ME-2), adenylate kinase(AK-1)
ex) mammal¿¡¼ µÎ°¡ÁöÀÇ lactate dehydrogenase activity ³ªÅ¸³»´Â µÎ°¡Áö typeÀÇ polypeptide°¡ ÀÖ´Ù.
* heart tissue¿¡ Ư»ö : H, muscle tissue¿¡ Ư»ö : M --> ÀÌ°ÍÀº tertamer Çü¼º À§ÇØ associateÇÑ´Ù. --> cell¿¡¼ ¹ß°ßµÇ´Â enzymeÀÌ´Ù.
4.4.7. Identifying the stage of differentiation
* tissue culture¿¡¼ cellÀ» divideÇÏ°íÀÚ ÇÏÁö¸¸, tissue´Â Á¤»óÀûÀ¸·Î proliferateÇÏÁö ¾Ê´Â fully differentiated cellº¸´Ù ¿ÀÈ÷·Á precusor cellÀ» representÇÑ´Ù. (mature differentiated cellÀº divideÇÏÁö ¾Ê´Â´Ù.)
* in vivoÀÇ cellÀº µ¿½Ã¿¡ divide ability¿Í ¾î¶² differentiated productÀÇ ¹ßÇöÀ» ÇÏÁö ¾Ê´Â´Ù. ¿©ÀüÈ÷ divide ability¸¦ °¡Áö´Â cell lineÀº differentiated properties¸¦ ¿ÏÀüÈ÷ ¹ßÇöÇÏÁö ¾Ê´Â´Ù.
* culture¿¡´Â genetically homogeneous Çصµ pheonotypically heterogeneousÇÏ´Ù. differentiationÀÇ ´Ù¸¥ stageÀÇ stem cell, precursor cell, differentiated cellÀ» Æ÷ÇÔÇÑ´Ù.
¢¡correct enviroment¸¦ re-creating ÇÔÀ¸·Î¼ cellÀÇ pure population¿¡¼ differentiated phenotypeÀ» re-induceÇØ¾ß ÇÑ´Ù.
¡æ propagation°ú differentiationÀ» À§ÇØ ´Ù¸¥ conditionÀÌ ÇÊ¿äÇÏ´Ù.
cell ´ë·®Áõ½Ä ¡æ cell division stop ¡æ Àû´çÇÑ inducer¸¦ ÷°¡ÇÏ¿© ºÐÈÀ¯µµ ¡æ correct cell-cell À̳ª cell-extracellular matrix interactionÀ» creating
4.4.8. Re-introducing the differentiated state in vitro
* cell cultureÀÇ ±Ã±ØÀû ¸ñÀûÀÇ Çϳª´Â cell culture enviroment¸¦ ´Ù¸¥ in vitro state¿¡¼ manupulateÇϴµ¥ ÀÖ´Ù.
* terminal differentiationÀ» ÇâÇÑ cellÀÇ ´Ù¸¥ lineage¸¦ delay, direct, promoteÇÏ´Â factor¸¦ °áÁ¤ÇÏ´Â Á¶°Ç¿¡ ´ëÇÑ µµÀü
¡æ extracellular matrix, cell-cell interaction, soluble inducer ¡æ ºÐÈÃËÁø factor
a. extracullular matrix ( in vivo¿¡¼ ÀûÀýÇÑ substrate¿Í ºñ½ÁÇÑ ¿ªÇÒ)
natural growth substratumÀ» Çü¼º, in vivo¿¡¼ static structural support cell behaviour¿¡ ¿µÇâÀ» ¹ÌÄ¡´Â distinct molecule·Î ±¸¼º ¡æ ºÐÈÀ¯µµ ¿ä¼Ò
ex) mammary epithelial cellÀÇ ductal morphogenesis¿¡¼ collagen I Àº in virtro¿¡¼ extracellular matrixÀÇ ´Ù¾çÇÑ component¿¡ ÀÇÇØ differentiated state¿¡ ¿µÇâÀ» ³¢Ä§
b. cell-cell interaction ( in vivo¿¡¼ÀÇ cell to cell contact¿Í ºñ½Á)
homologous cell interactionÀº high cell density¿¡¼ ÃÖÀûÀ¸·Î ÀϾÙ.
¡æ gap junctional communication, surface information exchange
¢¡ normal developmenet µ¿¾È differentiationÀ» initiating°ú promoting ÇÑ´Ù.
c. soluble inducers (hormone-endocrine signaling in vivo¿Í ºñ½Á)
differentiationÀÇ physiological inducer ¡æ in vitro¿¡¼ ºÐÈÀ¯µµ
hormone, paracrine factor, vitamin A, inorganic ion(Ca 2+)
ex) DMSO µµ ¾î¶² cell lineÀÇ ºÐȸ¦ À¯µµÇÑ´Ù.
4.5. Microbial contaminations
4.5.1. Bacteria, fungi, yeast
# contaminationÀÌ µÇ¸é ½±°Ô ±¸ºÐÇÒ ¼ö ÀÖ´Ù.
¡æ pHÀÇ ºü¸¥ º¯È(pheonol redÀÇ colorº¯È·Î ±¸ºÐ), mediumÀÇ cloudiness, extracellular granularity, medium¿¡ unidefined materialÀÇ floating
# °áÁ¡ÀÇ º¸¿Ï ¡æ ¹èÁö¿¡ Ç×»ýÁ¦ ÷°¡
* Gram positive organismÀÇ contamination ¡æ penicillin »ç¿ë
* Gram negative organismÀÇ contamination ¡æ streptomycin »ç¿ë
* fungi contamination ¡æ amphotericin B ³ª hystatin »ç¿ë
¢¡ cocktail·Î ¸¸µé¾î medium¿¡ ÷°¡ÇÑ´Ù.
4.5.2. Mycoplasma
# Å©±â°¡ 0.3§º¸´Ù ÀÛ¾Æ º¸ÀÌÁö ¾Ê´Â´Ù.
# self-replicating organism, contaminated cell¿¡ ¾î¶² overt change ¾øÀÌ proliferateÇÑ´Ù.
# cellular biochemistry, antigenicity, growth chracteristics¿¡ ½É°¢ÇÏ°Ô ¿µÇâÀ» ÁØ´Ù.
# media, sera, trypsin, operator·ÎºÎÅÍ infected µÊ
# ÁÖ±âÀûÀ¸·Î mycoplasma test ÇÊ¿ä
# ½Äº°¹æ¹ý
mycoplasma DNA´Â Hoechst 33258 fluorescent dye(human cell¿¡´Â bind ¾ÈÇÔ)¿¡ bindÇÑ´Ù.
¡æ ÀÌ dye¿¡ stain µÈ cellÀº fluorescent nuclei¸¦ °¡Áü ¡æ UV light¿¡¼ ¹ß±¤
* negative control : cell test°¡ Ãß°¡µÇÁö ¾Ê´Â control
* positive control : ¾Ë·ÁÁø mycoplasma¸¦ °íÀÇÀûÀ¸·Î Ãß°¡ÇÑ °ÍÀ» control
¡Å test sampleÀ» 8 ~ 12 hr incubate ¡æ methanol : acetic acid (v/v 3:1)·Î fixed ¡æ air dired ¡æ Hoechst 33258·Î stained ¡æ UV incident light »ç¿ëÇÏ¿© ÃøÁ¤ ¡æ small fluorescent filament³ª cocci°¡ Á¸ÀçÇϸé mycoplama¸¦ ÁöĪÇÑ´Ù.
# DNA stain
[ Bethesda Research Laboratory market system]
¨ç MycoTect
cellÀ» 6-methyl purine deoxyriboside(non-toxic to mammalian cell)·Î incubate ¢¡ mycoplasma¿¡ ÀÇÇØ 6-methyl purine deoxyriboside°¡ metaboliseµÇ¾î 6-methyl purine riboside(toxic to mammalian cell)·Î º¯ÇÑ´Ù ¢¡ ±×·¯¹Ç·Î, mycoplasma°¡ ÀÖ´Â mammalian cellÀº Á×°Ô µÈ´Ù
¨è Gene-probe
mycoplasma DNA¿Í hybridiseµÇ´Â ©øH-labelled DNA probe(mammalian DNA¿Í´Â bindÇÏÁö ¾Ê´Â´Ù)¸¦ »ç¿ë
¡æ electrophoresis·Î ½±°Ô ºÐ¸® °ËÃâÀÌ °¡´ÉÇÏ´Ù.
4.5.3. Viruses
persistent³ª latent virus infectionÀÌ cell line¿¡ Á¸ÀçÇϸé, immunological, cytological ultrastructural, biochemical test°¡ ¼öÇàµÇ±â±îÁö ¹ß°ßÇÏÁö ¸øÇÑ´Ù.
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