Laserova zaâ chytovaâ mikrodisekce ve vyâ zkumu odontogeneze Laser capture microdissection in odontogenesis research

Transcription

1 rocïnõâk19 ORTODONCIE Laserova zaâ chytovaâ mikrodisekce ve vyâ zkumu odontogeneze Laser capture microdissection in odontogenesis research *, **doc. RNDr. Eva MatalovaÂ, Ph.D., ***MUDr. PrÏemysl KrejcÏ õâ, Ph.D., *, **prof. MVDr. Ivan MõÂsÏ ek, CSc. *LaboratorÏ embryologie zï ivocï ichuê,uâ stav zï ivocïisïneâ fyziologie agenetiky, AV CÏ R v.v.i., Brno *Laboratory of Animal Embryology, Institute of Animal Physiology and Genetics, v.v.i., AS CR Brno **Veterina rnõâ a farmaceutickaâ univerzitabrno **University of Veterinary and Pharmaceutical Sciences Brno ***KlinikazubnõÂho leâkarïstvõâ, LeÂkarÏska fakulta, Univerzita Palacke ho, Olomouc ****Clinic of Dental Medicine, Medical Faculty, Palacky University Olomouc Souhrn Komplex vyvõâjejõâcõâho se zubu a okolnõâch struktur zahrnuje rïadu typuê diferencovanyâ ch buneïk.tysecï asto vyskytujõâ ve velmi malyâ ch pocï tech, ale majõâ velkyâ biologickyâ vyâ znam v morfogenezi (naprï. signaâ lnõâ centra sklovinnyâ ch uzluê ). MozÏnost izolace homogennõâch vzorkuê z konkreâ tnõâch tkaânï oveï vaâ zanyâ ch buneïcïnyâ ch populacõâ byla dlouhou dobu omezena. Nova technologie systeâ mu laseroveâ zaâ chytoveâ mikrodisekce (LCM) zprostrïedkovaâ vaâ rïadu prïesnyâ ch vyâ zkumuê na buneï cï neâ a subbuneï cï neâ uâ rovni. V poslednõâm desetiletõâ bylo techniky LCM vyuzï ito takeâ pro neï ktereâ studie zameïrïeneâ na mechanismy vyâ voje zubuê a souvisejõâcõâ poruchy. CõÂlem tohoto cï laâ nku je strucï neâ shrnutõâ prïõâstupu LCM, jeho aplikacõâ ve vyâ zkumu odontogeneze i nejaktuaâ lneï j- sï õâch modifikacõâ kombinujõâcõâch LCM a pruê tokovou cytometrii pro analyâ zy jednotlivyâ ch buneï k na uâ rovni nukleovyâ ch kyselin a proteinuê (Ortodoncie 2010, 19, cï. 5, s ). Abstract Complex of developing tooth and surrounding structures comprises several types of differentiated cells. They often occur in very small numbers but with a high biological impact on morphogenesis (e. g. signalling centres of enamel knots). However, isolation of specific homogenous samples from distinct tissue bound cell populations was long time limited. With appearance of novel technologies, the laser capture microdissection (LCM) system mediates many exact investigations at the cellular and subcellular level. In the last decade, the LCM technique has been used also for several studies focused on mechanisms of tooth development and related disorders. This paper aims to briefly review the LCM approach, its application in odontogenesis research, and the most recent modification combining LCM and flow cytometry for single cell analysis at nucleid acid and protein levels (Ortodoncie 2010, 19, No. 5, p ). KlõÂcÏ ovaâ slova: Laserova zaâ chytovaâ mikrodisekce Key Words: Laser capture microdissection Zuby se vyvõâjejõâ nazaâ kladeï reciprokyâ ch buneï cï nyâ ch amolekulaâ rnõâch signaâ luê mezi epitelem aprïileâ hajõâcõâm mesenchymem [23]. V pruêbeï hu odontogeneze se formujõâ specifickeâ signaâ lnõâ buneï cï neâ populace znaâ meâ jako sklovinneâ uzly [1]. BeÏ hem dalsï õâ diferenciace dochaâzõâ Teeth develop on basis of reciprocal cellular and molecular signalling between the epithelium and the underlying mesenchyme [23]. In the course of odontogenesis, specific signalling cell populations known as the enamel knots [1]. During further differentiation, stratum interme redakce@orthodont-cz.cz

2 ORTODONCIE rocïnõâk19 dium, reticulum stellate and inner dental epithelium becomes integrated from the epithelial part and the future tooth pulp organizes from the mesenchymal part of the tooth germ. On the border of both layers, dentin secreting odontoblasts differentiate from the mesenchyme and enamel producing ameloblasts from the epithelium. During odontogenesis, the tooth germ integrates with the surrounding tissues to form afunctional tooth-bone complex [24]. To study particular single cells or specific populations bound in tissues, the methods of choice were limited mostly to histology, histochemistry, immunohistochemistry and in situ hybridizations. Other methods for gene expression studies in samples from bones, tooth germs and other tissues were complicated by the fact that the specimen collected for analysis often represents a wide variety of cells distinct in individual phenotype, age, and state of maturation. The repertoire of techniques for precise cell analyses of tissue bound populations has been revolutionized by the introduction of laser capture microdissection (LCM) [2]. LCM, as a modified microscopic technique (Fig. 1a), allows for selection of specific cells or cell populations within histological sections followed by contact-free catapulting of the sample into a test-tube lid for further prok integraci stratum intermedium, reticulum stelatum avnitrïnõâho zubnõâho epitelu z epiteliaâ lnõâ cï aâ sti zubnõâho zaâ kladu, zatõâmco mesenchymaâ lnõâ cï aâ st daâvaâ vznik zubnõâ pulpeï. Narozhranõ obou vrstev se diferencujõâ mesenchymaâ lnõâ, dentin sekretujõâcõâ odontoblasty a epiteliaâ lnõâ, sklovinu tvorïõâcõâ ameloblasty. V pruêbeï hu odontogeneze se zubnõâ zaâ klad integruje s okolnõâmi tkaâ neï miza vzniku funkcï nõâho komplexu zubu akosti [24]. Metody pro studium konkreâ tnõâch jednotlivyâ ch buneï k nebo specifickyâch populacõâ vaâ zanyâch ve tkaânõâch byly omezeny nahistologii, histochemii, imunohistochemii a in situ hybridizace. Jine metody pro studium genoveâ exprese ve vzorcõâch kostõâ, zubnõâch zaâ kladuê adalsï õâch tkaâ nõâ byly komplikovaâ ny skutecï nostõâ, zïe vzorek odebranyâ pro analyâzu cï asto zahrnoval smeï s buneï k ruê znyâ ch fenotypuê, staâ rïõâ astaâ dia maturace. Revoluci v repertoaâ ru technik pro prïesneâ buneï cï neâ analyâ zy z tkaâ nï oveï vaâ zanyâ ch populacõâ znamenalo zavedenõâ laseroveâ zaâ chytoveâ mikrodisekce (LCM) [2]. LCM jako modifikovanaâ mikroskopickaâ technika(obr. 1a) umozïnï uje vyâbeï r jednotlivyâch buneï k nebo buneïcïnyâch populacõâ v raâ mci histologickeâ horïezu, kteryâ je naâ sledovaâ n bezkontaktnõâm katapultem vzorku do võâcï kazkumavky pro dalsï õâ zpracovaâ nõâ (Obr. 1b). StrucÏ neï shrnuto, nejprve je barvenyâ nebo nebarvenyâ histologickyâ rïez na beïzïneâ m nebo membraâ nou kryteâ m sklu umõâsteï n pod LCM mikroskop. V dalsï õâm kroku je vyuzïõâvaâ no zobrazenõâ zorneâ ho pole s tkaâ nõâ naobrazovce pocï õâta cï e, kde jsou pohybem mysï i zakresleny bunï ky pro vyâbeï r. Pote je nad rïez umõâsteïnovõâcï ko zkumavky naplneïneâ vodou, pufrem nebo jinou tekutinou. V dalsï õâm kroku je prïesneï podle zakresleneâ linie proveden laserovyâ rïez (Obr. 2). RÏ ez muêzïe byât ihned naâ sledovaâ n katapultem nebo lze katapult nastavit nezaâ visle pozdeï ji. TõÂmto zpuê sobem je vybranaâ oblast tkaâneï vystrïelenajako jeden kus. ObdobneÏ mohou byâ t katapultovaâ ny jednotliveâ body. Obr. 1a. Syste m laseroveâ zaâ chytoveâ mikrodisekce (Olympus). CÏ erveneï vyznacï enyâ vyârïez je v detailu popsaâ n naobraâ zku 1b. Fig. 1a. Laser capture microdissection system (Olympus). The red frame is described in detail in Fig. 1b. Obr. 1b. Nastavenõ LCM na selekci buneï k/tkaâ nõâ a jejich katapult do zkumavky. 1-laser, 2-opticka cesta, 3-koncentrovany laserovyâ paprsek, 4-konfoka lnõâ zesõâlenõâ (rovinavzorku), 5-mikroskopicke sklo se vzorkem, 6-smeÏ r umõâsteï nõâ zkumavky prïed katapultem (musõâ byât nad vzorkem), 7-võÂcÏ ko zkumavky na zaâ chyt katapultovaneâ ho vzorku. Fig. 1b. Setting of LCM for cell/tissue selection and folowing catapult into the test tube. 1-laser, 2-optical pathway, 3-concentrated laser beam, 4-confocal volume (sample level), 5-microscopic slide with the sample, 6-way of positioning the test tube prior to catapult (must be above the sample), 7-test tube lid for selected catapulted sample capture. redakce@orthodont-cz.cz 37

3 rocïnõâk19 ORTODONCIE V obou prïõâstupech je vzorek lokalizovaâ n ve võâcï ku zkumavky, odkud je centifugacõâ prïemõâsteï n najejõâ dno pro dalsï õâ zpracovaâ nõâ. V prïõâpadeï veï tsï õâch barvenyâ ch vzorkuê lze pomocõâ kameroveâ ho systeâ mu snadno oveï rïit jejich prïõâtomnost ve võâcï ku zkumavky. Ve stomatologickeâ mvyâ zkumu zahrnuje vyuzï itõâ LCM zejmeâ nasledovaâ nõâ genoveâ exprese in vivo zalozï eneâ na izolaci mrna naâ sledovaneâ reverznõâ transkripcõâ a polymeraâ zovou rïeteï zovou reakcõâ. Jednotlive bunï ky jako naprï. odontoblasty zõâskaneâ LCM zuê staâvaâ jõâ chemicky nezmeï neï ny, cï õâmzï jsou umozïneï ny prïesneâ molekulaâ rnõâ abiochemickeâ analyâ zy [3, 4]. S vyuzï itõâm LCM byladokumentovaâ na exprese PTHrP (parathyroid hormonerelated protein) v reticulum stellatum [5, 6]. V mikrodisekovanyâ ch vzorcõâch bylaprovaâ deï na kvantitativnõâ analyâ zalamininu aintegrinu nauâ rovni mrna zauâ cï e- lem zhodnocenõâ mozï nyâ ch rolõâ teï chto molekul v adhezõâch [7] abylaukaâ zaâ na exprese kalpainu u mysï õâch molaâ ruê [8]. LCM bylavyuzï itatakeâ pro genovou analyâ zu, zejmeâ na TGF-beta u potkana, a to v periodontaâ lnõâm ligamentu, charakteristickeâ populaci buneï k, kterou je vsï ak obtõâzïneâ zõâskat prïõâmo [9, 10]. Exprese TGF-beta bylazkoumaâ na take u mikrodisekovanyâ ch vzorkuê vazivaperiodontaâ lnõâho aparaâ tu aprïi studiu mechanismuê aktivnõâ erupce molaâ ruê [10]. LCM byladaâ le vyuzï itaprïi detekci exprese IL-10 v zubnõâm folikulu u potkanuê [11], CSF-1 v ameloblastech a odontoblastech osteopetrotickyâch (op/op) mysï õâ pro odhalenõâ op/op mutace naformovaâ nõâ primaâ rnõâ zubnõâ matrix [12]. Take molekuly souvisejõâcõâ s vyâ vojem kostõâ jako je RANKL [24] byly stu- cessing (Fig. 1b). Briefly summarized, the stained or unstained histological section is placed on a common or membrane coated slide under the LCM microscope in the first step. The following step uses the display of the visual field with the tissue on a computer screen to gate the cells of interest by a line made by the mouse movement. Afterwards, the test tube lid filled with water, buffer or other suitable liquid becomes positioned above the section. The next step is a laser cut exactly performed on the drawn line (Fig. 2). The cut can be immediately followed by a catapult or the catapult can be set independently later. This way, the selected tissue area becomes catapulted in one piece. Alternatively, the area can be catapulted as individual spots. In both approaches, the sample is located in the test tube lid to be centrifuged into the tube and further processed. In the case of larger stained samples, the presence of the catapulted area in the lid can be easily checked using a camera system. In dental research, the exploitation of LCM covers particularly in vivo gene expression investigation based on mrna isolation followed by reverse transcription and polymerase chain reaction. Individual cells, such as odontoblasts obtained by LCM are chemically unaltered, allowing accurate molecular and biochemical analyses [3, 4]. Using LCM, PTHrP (parathyroid hormone-related protein) expression was documented in the stellate reticulum [5, 6]. Quantitative analysis of laminin and integrin mrna from microdissected samples was performed to evaluate possible roles of these molecules in adhesion [7] and the expression of calpains in mouse molar was Obr. 2. Katapult vybraneâ buneï cï neâ populace metodou LCM na prïõâkladu duktuê mysï õâ slinneâ zïlaâ zy. Cely proces je rïõâzen naobrazovce pocï õâta cï e. 1-vzorek barvenyâ hematoxylinem a eosinem umõâsteï nyâ napodlozï nõâm skle pokryteâ m termostabilnõâ membraâ nou, 2-vyÂbeÏ r oblasti pro katapult kresbou naobrazovce pocïõâtacï e, 3-katapult vybraneâ oblasti (na skle zuê stane odpovõâdajõâcõâ praâ zdnyâ prostor). Fig. 2. Catapult of selected cell population by the LCM method, an example of mouse salivary gland ducts. The entire procedure is controlled on the computer screen. 1-sample stained by hematoxylin-eosin placed on a thermostabile membrane coated slide, 2- selection of area of intest on the computer screen, 3-catapult of the selected area (corresponding empty space appears on the slide) redakce@orthodont-cz.cz

4 ORTODONCIE rocïnõâk19 dovaâ ny v mikrodisekovanyâ ch vzorcõâch ze zubnõâho folikulu [13, 14]. U poraneï nõâ zubuê bylasledovaâ nahladina exprese mrna u proteinuê asociovanyâ ch s tvrdyâ mi tkaâneï mi (osteopontin, osteonektin, osteokalcin) a alfa 2 deltapodjednotky napeï tõâm rïõâzeneâho vaâ pnõâkoveâ ho kanaâ lu (CACNA2D1) v apikaâ lnõâ cïaâ sti akoronaâ rnõâ cïaâ sti pulpy. ReverzneÏ transkripcï nõâ-polymeraâ zovaâ rïeteï zovaâ reakce (RT-PCR) byla provaâdeï nanalcm vzorcõâch [15]. BeÏ hem poslednõâho desetiletõâ bylo dosazï eno mnoha vyâ znamnyâ ch vyâ sledkuê praâ veï s vyuzï itõâm teâ to elegantnõâ techniky, ato zejmeâ nanauâ rovni nukleovyâ ch kyselin, kde je mozïnaâ amplifikace. OvsÏem v prïõâpadech, kdy amplifikacï nõâ metody nejsou dostupneâ, se hlavnõâ vyâhody LCM vytraâ cejõâ. Z tohoto duê vodu vyâsledky dosazï eneâ v proteomice zaostaâ vajõâ za genomickyâ mi atranskriptomickyâ mi studiemi [16, 17].Z duê vodu prïekonaâ nõâ obtõâzï õâ v zõâskaâ nõâ dostatecïneâ ho mnozï stvõâ tkaâ nõâ abuneïk pro proteinovou analyâ zu byl navrzï en novyâ prïõâstup zalo- shown [8]. LCM was used also in gene analysis, particularly TGF-beta in the rat periodontal ligament, a distinct population of cells, which is difficult to be obtained directly [9, 10]. TGF-beta expression was also examined in microdissected samples of the periodontal ligament to follow mechanisms of active eruption of molars [10]. LCM was further used to detect IL-10 gene expression in the rat dental follicle [11], CSF-1 in ameloblasts and odontoblasts of osteopetrotic (op/op) mice to see any impact of the op/op mutation on the primary tooth matrix formation [12]. Also bone related molecules, such as RA- NKL [24], were examined in laser microdissected samples from the dental follicle [13, 14]. In dental injuries, mrna expression levels of hard tissue-associated proteins (osteopontin, osteonectin, and osteocalcin) and calcium channel voltage-dependent alpha 2 delta subunit 1 (CACNA2D1) in the apical and coronal portions of the pulp. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on LCM samples [15]. Obr. 3. Kombinace LCM s pruê tokovou cytometriõâ pro analyâzu specifickyâch tkaânï oveï vaâ zanyâch buneïcïnyâch populacõâ na uâ rovni jednotlivyâch buneï k naprïõâkladu mysï õâho primaâ rnõâho sklovinneâ ho uzlu (PEK). Kryoprezervovane hlavoveâ cï aâ sti embryajsou zpracovaâ ny nafrontaâ lnõâ rïezy (25 mm), rïezy obsahujõâcõâ PEK jsou umõâsteï ny namembraâ nou krytaâ mikroskopickaâ sklapro LCM. 1-kompaktnõ vybranaâ populace buneï k PEK je katapultovaâ nado võâcï ka zkumavky, 2-kompaktnõ cï aâ st tkaâ neï je stocï enado zkumavky, 3-ve zkumavce jsou rozvolneï ny jednotliveâ bunï ky, 4-intaktnõ volneâ bunï ky mohou byât vyuzï ity pro libovolnou cytometrickou analyâzu, 4-po znacï enõâ jsou volneâ bunï ky vyhodnoceny pruê tokovyâ m cytometrem azastoupenõâ pozitivnõâch buneï k je vyjaâ drïeno v procentech (zde demonstrovaâ n vyâsledek TUNEL testu pro detekci apoptotickyâch buneï k). Fig. 3. Combination of LCM and flow cytometry for single cell analysis of specific tissue bound cell populations, an example of the mouse primary enamel knot (PEK). Cryopreserved embryonic heads are sectioned (25 mm), sections containing the PEK are placed on membrane coated slides for LCM. 1-compact selected cell population of the PEK is catapulted into the test tube lid, 2-compact tissue area is centrifuged into the test tube, 3- cells undergo individualization in the test tube, 4-intact individual cells can be used for any cytometric analysis, 4-labelled cells are evaluated by the flow cytometer and content of positive cells is expressed as percentage (here is shown a result of TUNEL test for detection of apoptotic cells). redakce@orthodont-cz.cz 39

5 rocïnõâk19 ORTODONCIE zï enyâ napruê tokoveâ cytometrii mikrodisekovanyâch vzorkuê [18]. Tato technika (LCM-FC) kombinuje vyâhody LCM, kteraâ poskytuje homogennõâ vzorky buneï k avyâ hody pruê tokoveâ cytometrie, kteraâ umozï nï uje hodnocenõâ jednotlivyâ ch buneï k. Tyto metody jsou propojeny enzymatickou a/nebo mechanickou desintegracõâ tkaâ nõâ. Vzorky jsou zõâskaâvaânyz25mm silnyâch kryorïezuê pro zajisïteï nõâ intaktnõâ strïednõâ vrstvy buneï k. Tento novyâ prïõâstup umozïnï uje analyâzu specifickyâch tkaânï oveï vaâzanyâ ch populacõâ, jako naprï. primaâ rnõâ sklovinnyâ uzel (PEK), nauâ rovni jednotlivyâch buneï k (Obr. 3). PEK se objevuje jako koncentricky usporïaâ danaâ skupinabuneï k ve strïedu zubnõâho pohaâ rku beï hem odontogeneze. PEK je obklopen epiteliaâ lnõâ cervikaâ lnõâ smycï kou, jejõâzï ruê st podporujõâpraâveï signaâ ly z nedeï lõâcõâho se PEK. TõÂmto zpuê sobem dosaâ hne zubnõâ zaâ klad tvaru zvonku, kdy je mesenchymaâ lnõâ papila zvolna obklopovaâ naepiteliaâ lnõâ cïaâ stõâ atak se objevuje zaâ kladnõâ forma pro budoucõâ mineralizovanyâ zub. Po splneï nõâ sveâ signaâ lnõâ funkce jsou bunï ky PEK eliminovaâ ny apoptoâ zou. Tyto nepocï etneâ specifickeâ bunï ky jsou vsï ak vysoce duê lezï iteâ pro zdaâ rnyâ vyâ voj dentice. LCM je metodou vyâ razneï napomaâ hajõâcõâ prïi zõâskaânõâcï isteâ populace PEK buneï k pro dalsï õâ stadium odontogeneze. Proteomicke analyâ zy jako SELDI (surface enhanced laser desorption/ionization) a MALDI (matrix assisted laser desorption/ionization) produkujõâ spolehliveâ vyâsledky jizï z neï kolikabuneï k, cozï cï inõâ tyto naâ rocï neâ prïõâstupy vhodnyâ mi pro hodnocenõâ LCM vzorkuê [19, 20]. BiomedicõÂnske aplikace mikrocï ipoveâ analyâ zy proteinuê [21] aktuaâ lneï otevrïely dalsï õâ alternativnõâ prïõâstup pro vyuzï itõâ LCM technologiõâ. Takove techniky jsou zalozï eny zejmeâ nanavyuzï itõâ extraktuê LCM zachyceneâ ho materiaâ lu, zatõâmco LCM-FC je zacõâlena na kompaktnõâ jednotliveâ bunï ky. MozÏ nosti aomezenõâ aktuaâ lneï pouzï õâvanyâch postupuê spojenyâ ch s hodnocenõâm LCM vzorkuê na uâ rovni proteinuê jsou staâ le diskutovaâ ny [22, 17]. AutorÏi nemajõâ komercï nõâ, vlastnickeâ nebo financï nõâ zaâ jmy na produktech nebo spolecï nostech popsanyâ ch v tomto cïlaâ nku. PodeÏ kovaâ nõâ Grantova podpora aktuaâ lnõâho vyâ zkumu: GA AV KJB (molekulaâ rnõâ odontogeneze), GA AV IAA (molekulaâ rnõâ embryogeneze), GA CÏ R 524/08/J032 (buneï cï neâ a molekulaâ rnõâ interakce beï hem vyâ voje zubuê a kostõâ), GA CÏ R 203/08/1680 (noveâ metodickeâ prïõâstupy). During the last ten years, many significant results have been achieved using this elegant technique, particularly at nucleic acid level where amplification methods are available. However, in cases where amplification is not available, the great benefits of LCM fade. Therefore, the achievements in proteomics lack behind genomic and transcriptomic studies [16, 17]. To overcome difficulties in obtaining sufficient quantities of tissues and cells for protein analyses, a novel approach based on flow cytometry of laser micro-dissected samples has been introduced [18]. The technique (LCM- FC) combines benefits of LCM providing homogenous samples of cells of interest and flow cytometry allowing for evaluation of individual cells. These methods are linked by enzymatic and/or mechanical tissue disintegration. Samples are obtained from 25 mm thick cryopreserved sections to guarantee an intact layer of cells in the middle. This novel approach allows for single cell analysis of specific tissue bound cell populations, such as the primary enamel knot (PEK) (Fig. 3). PEK appears as a concentrically arranged cell cluster at the heart of the tooth cap during prenatal odontogenesis. PEK is surrounded by epithelial cervical loop and its growth is supposted by signals from the non-dividing PEK. This way the tooth germ reaches the bell stage when the mesenchymal papilla becomes gradually wrapped by the epithelial part and thus the basic form for the final mineralized tooth appears. Therefore, the few but specific PEK cells are highly important for successful odontogenesis. After fulfilling its signalling mission, the PEK cells undergo apoptosis. LCM has greatly helped to access pure populations of PEK cells for further studies of odontogenesis. Proteomic analysis, such as SELDI (surface enhanced laser desorption/ionization) and MALDI (matrix assisted laser desorption/ionization), generate reliable results from as few as a dozen cells making these demanding approaches well suited for analysis of LCM samples [19, 20]. Biomedical application of protein microarray analysis [21] has recently opened another alternative approach to exploit LCM technology. These techniques are mostly based on extracts of LCM captured material, whereas LCM-FC targets compact individual cells. The possibilities and limitations of these recent procedures for protein evaluation in LCM samples have been still discussed [22, 17]. The authors have no comercial, proprietary, or financial interests in the products or companies described in this artikle. Acknowledgement Grant support of recent research: GA AV KJB (molecular odontogenesis), GA AV IAA (molecular embryogenesis), GA CR 524/08/J032 (cellular and molecular interactions during tooth-jawbone development), GA CR 203/08/ 1680 (new methodical approaches) redakce@orthodont-cz.cz

6 ORTODONCIE rocïnõâk19 Literatura/References 1. Jernvall, J.; Kettunen, P.; Karavanova, I.; Martin, L.B.; Thesleff, I.: Evidence for the role of the enamel knot as a control center in mammalian tooth cusp formation - non-dividing cells express growth-stimulating Fgf-4 gene. Int. J. dev. Biol. 1994, 38, s Simone, N.L.; Bonner, R.F.; Gillespie, J.W.; Emmert- Buck, M.R.; Liotta, L.A.: Laser-capture microdissection: opening the microscopic frontier to molecular analysis. Trends Genet. 1998, 14, s Hoffmann, M.; Olson, K.; Cavender, A.; Pasqualini, R.; Gaikwad, J.; D'Souza, R.: Gene expression in a pure population of odontoblasts isolated by laser-capture microdissection. J. dent. Res. 2001, 80, s McLachlan, J.L.; Smith, A.J.; Cooper, P.R.: Piezo-power microdissection of mature human dental tissue. Arch. oral Biol. 2003, 48, s Wise, G.E.; Ding, D.; Yao, S.: Regulation of secretion of osteoprotegerin in rat dental follicle cells. Eur. J. oral Sci. 2004, 112, s Yao, S.M.; Pan, F.H.; Wise, G.E.: Chronological gene expression of parathyroid hormone-related protein (PTHrP) in the stellate reticulum of the rat - Implication for tooth eruption. Arch. oral Biol. 2007, 52, s Kinumatsu, T.; Hashimoto, S.; Muramatsu, T.; Sasaki, H.; Jung, H.S.; Yamada, S.; Shimono, M.: Involvement of laminin and integrins in adhesion and migration of junctional epithelium cells. J. periodont. Res. 2009, 44, s Matsunaga, T.; Yamamoto, G.; Tachikawa, T.: Expression of typical calpains in mouse molar. Arch. oral Biol. 2009, 54, s Nakamura, Y.; Nomura, Y.; Arai, C.; Nodal, K.; Oikawal, T.; Kogurel, K.; Kawamoto, T.; Hanada, N.: Laser capture microdissection of rat periodontal ligament for gene analysis. Biotech. Histochem. 2007, 82, s Oikawa T.; Nomura Y.; Arai C.; Noda K.; Hanada N.; Nakamura Y.: Mechanism of active eruption of molars in adolescent rats. Eur. J. Orthodont. 2010, 22. [Epub ahead of print] 11. Liu, D.W.; Yao, S.M.; Wise, G.E.: Effect of interleukin-10 on gene expression of osteoclastogenic regulatory molecules in the rat dental follicle. Eur. J. oral Sci. 2006, 114, s Werner, S.A.; Gluhak-Heinrich, J.; Woodruff, K.; Wittrant, Y.; Cardenas, L.; Roudier, M.; MacDougall, M.: Targeted expression of cscsf-1 in op/op mice ameliorates tooth defect. Arch. oral Biol. 52, Yao, S.M.; Ring, S.; Henk, W.G.; Wise, G.E.: In vivo expression of RANKL in the rat dental follicle as determined by laser capture microdissection. Arch. oral Biol. 2004, 49, s Wise, G.E.; Yao, S.: Regional differences of expression of bone morphogenetic protein-2 and RANKL in the rat dental follicle. Eur. J. oral Sci. 2006, 114, s Kaneko, T.; Okiji, T.; Kaneko, R.; Sunakawa M.; Kaneko M.; Suda H.: Gene expression analysis of acutely traumatized pulps. J. Endod. 2010, 36, s Leary, J.F.; Szaniszlo, P.; Prow, T.; Reece, L.M.; Wang, N., Asmuth, D.M.: The importance of high-throughput cell separation technologies for genomics/proteomicsbased clinical diagnostics. Clin. Diag. Syst. 2002, 4625, s Murray, G.I.: An overview of laser microdissection technologies. ActaHistochem. 2007, 109, s Matalova, E.; Dubska, L.; Fleischmannova, J.; Chlastakova, I.; Janeckova, E.; Tucker, A.S.: Cell proliferation and apoptosis in the primary enamel knot measured by flow cytometry of laser microdissected samples. Arch. oral Biol. 2010, 55, s Bhattacharya, S.H.; Gal, A.A.; Murray, K.K.: Laser capture microdissection MALDI for direkt analysis of archival tissue. J. Proteom. Res. 2003, 2, s Espina, V.; Dettloff, K.A.; Cowherd, S.; Petricoin, E.F.; Lotta, L.A.: Use of proteomic analysis to monitor responses to biological therapies. Exp. Opin. Biol. Ther. 2004, 4, p Spisak, S.; Guttman, A.: Biomedical applications of protein microarrays. Cur. Med. Chem. 2009, 16, s Craven, R.A.; Banks, R.E.: Laser capture microdissection and proteomics: Possiblities and limitation. Proteomics 2001, 1, s FleischmannovaÂ, J.; KrejcÏ õâ, P.; MatalovaÂ, E.; MõÂsÏ ek, I.: Molekula rnõâ podstata vyâ voje zubnõâch zaâ kladuê. Ortodoncie 2007, 16, cï.4, s KrejcÏ õâ, P.; MatalovaÂ, E.; FleischmannovaÂ, J.; MõÂsÏ ek, I.: Molekula rnõâ souhramorfogeneze zubuê a cï elistnõâch kostõâ, souvisejõâcõâ poruchy. CÏ aâ st 2: Remodelace a defekty. Ortodoncie 2009, 18, cï.5, s Doc. RNDr. Eva MatalovaÂ, PhD. LaboratorÏ embryologie zï ivocï ichuê U ZÏ FG AV CÏ R, v.v.i. VeverÏõ 97, Brno CÏ lenskyâ poplatek pro rok 2011 cï inõâ 1500,- KcÏ nebo 65,- EUR. CÏ lenoveâ v zameï stnaneckeâ m vztahu 800,- KcÏ nebo 35,- EUR. Postgraduanti, duê chodci a zïeny na materïskeâ dovoleneâ 300,- KcÏ nebo 15,- EUR. RegistracÏ nõâ polatek cï inõâ 500,- KcÏ nebo 20,- EUR. PrÏedplatne cï asopisu Ortodoncie pro necï leny CÏ OSje 1000,- KcÏ za rok nebo 40,- EUR. U hrada poplatku do , cï.uâ.: /0100, konst. symbol: 0558, variab. symbol: rodneâ cï õâslo. PrÏi nezaplacenõâ prïõâspeï vkuê po dvou põâsemnyâch urgencõâch bude ukoncï eno cï lenstvõâ v CÏ OS. redakce@orthodont-cz.cz 41