Sinteza konjugatov lektin-koloidno zlato za lokalizacijo sladkornih preostankov v urotelijskih celicah

Grdadolnik, Maja (avtor), Zupančič, Daša (mentor)

Lektini so glikoproteini, ki specifično prepoznajo in se vežejo na sladkorne preostanke v celicah. Koloidno zlato je suspenzija nanometrskih delcev zlata v vodnem mediju, ki se uporablja kot označevalec v elektronski mikroskopiji. Lektine, konjugirane s koloidnim zlatom velikosti od 5 do 20 nm, lahko uporabljamo v presevni elektronski mikroskopiji za označevanje sladkornih preostankov v zdravih in patološko spremenjenih vzorcih. V magistrski nalogi smo uporabili tri metode za sintezo delcev koloidnega zlata velikosti 20 nm in 10 nm. Po citratni metodi CM-20nm-1 in obratni citratni metodi OCM-10nm smo uspešno sintetizirali koloidno zlato, po citratni metodi CM-20nm-2 pa se je koloidno zlato oborilo. Z lektinoma ACA in jakalin (Jac), ki specifično prepoznata galaktozil(β-1,3)N-acetilgalaktozamin smo konjugirali koloidno zlato, sintetizirano po metodah CM-20nm-1 in OCM-10nm. Uspešnost sinteze koloidnega zlata in konjugacije z lektinoma smo preverjali s presevnim elektronskim mikroskopom (TEM) ter spektrofotometrom za merjenje UV-VIS spektra (190 nm do 900 nm). Rezultati TEM in UV-VIS spektroskopije so pokazali, da so delci koloidnega zlata sintetizirani po metodah CM-20nm-1 in OCM-10nm veliki okoli 16 nm. Uspešnosti konjugacije s TEM nismo mogli potrditi, s UV-VIS spektroskopijo pa smo potrdili, da je bila konjugacija lektinov ACA in Jac s koloidnim zlatom uspešna. Čeprav nam ni uspelo sintetizirati koloidnega zlata različnih velikosti, smo uspeli pokazati, da sta postopek sinteze koloidnega zlata velikosti okoli 16 nm in nadaljnja konjugacija z lektinoma ACA in Jac enostavna in ponovljiva. Na parafinskih rezinah mišjega sečnega mehurja in korteksa ledvic smo izvedli lektinsko histokemijo s fluorescenčno označenimi lektini (ACA-FITC in Jac-FITC), ki so pokazali vezavo na obrobne dele urotelijskih celic ter na distalne tubule in glomerule korteksa ledvic. Tkivo mišjega korteksa ledvic smo zato izbrali za pozitivno kontrolo, ker sta se konjugata ACA-FITC in Jac-FITC specifično vezala na glomerule in distalne tubule. Da bi lahko ugotovili ultrastrukturno lokalizacijo sladkornih preostankov v urotelijskih celicah, smo izvedli lektinsko histokemijo na ultratankih rezinah mišjega sečnega mehurja. Uporabili smo različne načine kemijske fiksacije (4-odstotni FA/PHEM ali 1-odstotni GA/PHEM) in vklapljanja tkiva sečnega mehurja ter primerjali specifičnost vezave komercialno dostopnih konjugatov lektin-koloidno zlato s konjugati lektin-koloidno zlato, ki smo jih sintetizirali. Ugotovili smo, da je vezava sintetiziranih in komercialno dostopnih konjugatov na krioultratankih rezinah in ultratankih rezinah, vklopljenih v umetno smolo Lowicryl K4M, nespecifična. Vzrok za nespecifično vezavo je najverjetneje sprememba sladkornih preostankov, ki je posledica vklapljanja v umetno smolo Lowicryl K4M oziroma v želatino pri pripravi krioultratankih rezin. Ta sklep potrjujejo tudi rezultati pozitivne kontrole, saj se lektina ACA in Jac specifično vežeta na parafinske rezine (kemijska fiksacija s 4-odstotnim FA/PHEM), nasprotno pa je vezava na krioultratanke rezine (kemijska fiksacija s 4-odstotnim FA/PHEM ali z 1-odstotnim GA/PHEM) korteksa ledvic nespecifična.

• Jezik: Slovenski jezik
• Ključne besede: koloidno zlato, lektin, lektinska histokemija, presevna elektronska mikroskopija
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2019
• PID: 20.500.12556/RUL-110201
• COBISS.SI-ID: 34490585
• Datum objave v RUL: 12.09.2019

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Lectin-colloidal gold conjugate synthesis far localization ar sugar residues in urothelial cells

Izvleček

Lectins are (glycol)proteins with specific affinity to sugar residues in tissues and cells. Colloidal gold is a (water) suspension of gold nanoparticles. Lectin-colloidal gold conjugates can be used in transmission electron microscopy to label sugar residues in healthy and pathologically modified specimens. In the thesis we used three methods of synthesis of 20 nm and 10 nm colloidal gold particles. Colloidal gold was successfully synthesized by the CM-20nm-1 citrate method and OCM-10nm inverse citrate method, however by CM-20nm-2 citrate method the colloidal gold precipitated. We conjugated lectins ACA and jacalin (Jac), which specifically recognize galactosyl-(β-1,3)-N-acetylgalactosamine, with colloidal gold synthesized by using CM-20nm-1 and OCM-10nm methods. The results of colloidal gold synthesis and conjugation with lectins was verified by transmission electron microscopy (TEM) and a spectrophotometer to measure the UV-VIS spectrum (190 nm to 900 nm). The results of TEM and UV-VIS spectroscopy showed that the colloidal gold particles were synthesized by the CM-20nm-1 and OCM 10nm methods about 16 nm in size. The success of conjugation with TEM could not be confirmed, and UV-VIS spectroscopy confirmed that the conjugation of ACA and Jac lectins with colloidal gold was successful. Although we were unable to synthesize colloidal gold of different sizes, we were able to demonstrate that the process of colloidal gold synthesis of about 16 nm in size and subsequent conjugation with ACA and Jac lectins were simple and reproducible. We performed lectin histochemistry using lectins conjugated with fluorochromes (ACA-FITC and Jac-FITC) on paraffine sections of mouse urinary bladder and kidney renal cortey. The results showed specific labelling to the marginal parts of the urothelial cells and to the distal tubules and glomeruli of the renal cortex. Therefore, the mouse renal cortex tissue was selected for positive control. To determine the ultrastructural localization of the sugar residues in the urothelial cells, we performed lectin histochemistry on ultrathin sections of mouse bladder. Different chemical fixation methods (4-percent FA/PHEM and 1-percent GA/PHEM) and bladder tissue embedding techniques were used and the binding specificity of commercially available lectin-colloidal gold conjugates was compared with the lectin-colloidal gold conjugates that were synthesized. We found that the binding of the synthesized and commercially available conjugates on cryoultrathin sections and ultrathin sections embedded into the Lowicryl K4M artificial resin was nonspecific. This is probably due to the change in the sugar residues, which is the result of embedding into the Lowicryl K4M artificial resin or gelatin in the process of preparation of cryoultrathin sections. This conclusion was also confirmed by the results of the positive control where lectins ACA and Jac specifically bound to paraffin sections (chemical fixation with 4-percent FA/PHEM). In contrast, binding to cryoultrathin sections (chemical fixation with 4-percent FA/PHEM or 1-percent GA/PHEM) of renal cortex was nonspecific.

• Ključne besede: colloidal gold, lectin, lectin histochemistry, transmission electron microscopy