12 research outputs found
Multi-layered molecular mechanisms of polypeptide holding, unfolding and disaggregation by HSP70/HSP110 chaperones.
Members of the HSP70/HSP110 family (HSP70s) form a central hub of the chaperone network controlling all aspects of proteostasis in bacteria and the ATP-containing compartments of eukaryotic cells. The heat-inducible form HSP70 (HSPA1A) and its major cognates, cytosolic HSC70 (HSPA8), endoplasmic reticulum BIP (HSPA5), mitochondrial mHSP70 (HSPA9) and related HSP110s (HSPHs), contribute about 3% of the total protein mass of human cells. The HSP70s carry out a plethora of housekeeping cellular functions, such as assisting proper de novo folding, assembly and disassembly of protein complexes, pulling polypeptides out of the ribosome and across membrane pores, activating and inactivating signaling proteins and controlling their degradation. The HSP70s can induce structural changes in alternatively folded protein conformers, such as clathrin cages, hormone receptors and transcription factors, thereby regulating vesicular trafficking, hormone signaling and cell differentiation in development and cancer. To carry so diverse cellular housekeeping and stress-related functions, the HSP70s act as ATP-fuelled unfolding nanomachines capable of switching polypeptides between different folded states. During stress, the HSP70s can bind (hold) and prevent the aggregation of misfolding proteins and thereafter act alone or in collaboration with other unfolding chaperones to solubilize protein aggregates. Here, we discuss the common ATP-dependent mechanisms of holding, unfolding-by-clamping and unfolding-by-entropic pulling, by which the HSP70s can apparently convert various alternatively folded and misfolded polypeptides into differently active conformers. Understanding how HSP70s can prevent the formation of cytotoxic protein aggregates, pull, unfold, and solubilize them into harmless species is central to the design of therapies against protein conformational diseases
Εργαστηριακή διερεύνηση παιδιών με σύνδρομο Williams
Το σύνδρομο Williams (ΣW) είναι μια πολύ γνωστή νευροαναπτυξιακή διαταραχή που έχει επιπολασμό 1 / 7500-20000 ζωντανών γεννήσεων και προκύπτει κυρίως από ένα de novo έλλειμμα στην περιοχή 7q11.23 με μήκος 1,5 -1,8 Mb. Η αναδρομική μελέτη των ανήλικων ασθενών που παραπέμφθηκαν και εκτιμήθηκαν με κλινική εικόνα πιθανού ΣW από τις κλινικές γενετικής του Εργαστηρίου Ιατρικής Γενετικής του Πανεπιστημίου Αθηνών, με έδρα το Χωρέμειο Ερευνητικό Εργαστήριο στο Νοσοκομείο Παίδων «Αγία Σοφία» μεταξύ των ετών 2009-2016 καθώς και η ανασκόπηση της βιβλιογραφίας για το συγκεκριμένο σύνδρομο είναι το θέμα της μεταπτυχιακής μου εργασίας. Σκοπός είναι ο προσδιορισμός των πιο κοινών φαινοτυπικών γνωρισμάτων του συνδρόμου στον ελληνικό πληθυσμό αλλά και ο καθορισμός των αποτελεσματικότερων γενετικών εργαστηριακών τεχνικών που χρησιμοποιούνται ευρέως για την επιβεβαίωση της κλινικής διάγνωσης παιδιών με σύνδρομο Williams.
Έγινε σύγκριση των διαθέσιμων μέχρι σήμερα μοριακών κυτταρογενετικών τεχνικών και της εργαστηριακής ανάλυσης του DNA και συμπερασματικά η χρήση της τεχνικής του φθορίζοντα in-situ υβριδισμού (FISH) είναι, έως σήμερα, ο χρυσός κανόνας για τη διάγνωση του ΣW. Η μοριακή τεχνική MLPΑ θεωρείται πολύ αξιόπιστη με χαμηλό κόστος και ισάξια αποτελεσματικότητα σε σύγκριση με την ανάλυση χρωμοσωμικών μικροσυστοιχιών (chromosomal microarray analysis- CMA). Νέες μέθοδοι που χρησιμοποιούν μονονουκλεοτιδικές αλλαγές είναι τώρα διαθέσιμες για ευρεία χρήση ως δείκτες μικροδορυφορικών αλληλουχιών και βοηθούν να προσδιοριστεί τόσο το μέγεθος όσο και τη γονική προέλευση του ελλείματος στο ΣW, το οποίο είναι εξίσου σημαντικό για την περαιτέρω ανάπτυξη της ήδη υπάρχουσας γνώσης για αυτό το γενετικά σύνθετο σύνδρομο.
Τα κοινά φαινοτυπικά χαρακτηριστικά των ασθενών που διαγνώστηκαν με ΣW στο εργαστήριο είναι τρία: το χαρακτηριστικό προσωπείο του συνδρόμου (elfin face), η νοητική καθυστέρηση και η συγγενής καρδιοπάθεια, ευρήματα που συμβαδίζουν με την διεθνή βιβλιογραφία. Επί του παρόντος, δεν υπάρχει γενετική εξέταση που να προβλέπει τη σοβαρότητα του φαινοτύπου του συνδρόμου Williams σε έναν συγκεκριμένο ασθενή. Περισσότερες μελέτες πρέπει να επικεντρωθούν σε έναν συσχετισμό φαινοτύπου-γονότυπου και να αποκαλύψουν πιθανές υποκείμενες επιγενετικές επιδράσεις που δεν έχουν αποδοθεί προς το παρόν στο μηχανισμό πρόκλησης του συνδρόμου.Williams syndrome (WS) is a well-known neurodevelopmental disorder that has a prevalence of 1/7500–20000 live births and results principally from a de novo deletion in 7q11.23 with a length of 1.5 -1.8 Mb. Thε retrospective data collection and analysis of the children that were referred and examined by the clinical geneticists with possible phenotype of WS in the Medical Genetics Laboratory of the University of Athens, based at the Choremio Research Laboratory of Aghia Sophia Children’s hospital between 2009-2016 as well as the literature review of WS is my master’s thesis project. It aims to determine the most common phenotypic characteristics of the syndrome in the Greek population and define the effectiveness of the existing laboratory investigations used to confirm the clinical diagnosis of children with WS.
Through comparing the available to date molecular cytogenetic techniques and DNA analysis laboratory testing, I would conclude that fluorescence in situ hybridisation (FISH) is the gold standard method used for the WS diagnosis. Multiplex ligation-dependent probe amplification (MLPA) is found to be very reliable and cost effective compared to array based comparative genomic hybridization (aCGH). New methods using nucleotide polymorphisms are now available to be used broadly as microsatellite markers and will help us determine both the size and parental origin of the deletion in WS, which is equally important in elaborating further our already existing knowledge of this genetically complex syndrome.
The common characteristics of the patients diagnosed with WS through our laboratory are three: the elfin face, mental retardation and congenital cardiac defects, findings that are in line with the existing literature. Currently, there is no genetic test that predicts the severity of the Williams syndrome phenotype in a given patient. More studies need to be focused towards a phenotype -genotype correlation and reveal possible underlying epigenetic effects that are not currently linked with the syndrome
Computing Network of Diseases and Pharmacological Entities through the Integration of Distributed Literature Mining and Ontology Mapping
The proliferation of -omics (such as, Genomics, Proteomics) and -ology (such as, System Biology, Cell Biology, Pharmacology) have spawned new frontiers of research in drug discovery and personalized medicine. A vast amount (21 million) of published research results are archived in the PubMed and are continually growing in size. To improve the accessibility and utility of such a large number of literatures, it is critical to develop a suit of semantic sensitive technology that is capable of discovering knowledge and can also infer possible new relationships based on statistical co-occurrences of meaningful terms or concepts. In this context, this thesis presents a unified framework to mine a large number of literatures through the integration of latent semantic analysis (LSA) and ontology mapping. In particular, a parameter optimized, robust, scalable, and distributed LSA (DiLSA) technique was designed and implemented on a carefully selected 7.4 million PubMed records related to pharmacology. The DiLSA model was integrated with MeSH to make the model effective and efficient for a specific domain. An optimized multi-gram dictionary was customized by mapping the MeSH to build the DiLSA model. A fully integrated web-based application, called PharmNet, was developed to bridge the gap between biological knowledge and clinical practices. Preliminary analysis using the PharmNet shows an improved performance over global LSA model. A limited expert evaluation was performed to validate the retrieved results and network with biological literatures. A thorough performance evaluation and validation of results is in progress
Aggregation of polyQ-expanded huntingtin interferes with cytosolic protein quality control by sequestering critical chaperone factors
Characterisation of Trypanosomal Type III and Type IV Hsp40 proteins
The heat shock protein-70 (Hsp70) family of molecular chaperones are ubiquitous highly conserved proteins that are critical for the viability of cellular homeostasis. The ATPase activity of Hsp70 proteins is critical to their function as the affinity of a given Hsp70 for non-native substrate is modulated by ATP binding and hydrolysis. When bound to ATP, Hsp70s possess a low affinity for a given substrate protein, while the hydrolysis of ATP to ADP causes a conformational change that results in a high affinity for substrate proteins. The basal ATPase activity of Hsp70s is too low to facilitate their function in vivo, and co-chaperones are essential to modulate the efficient protein folding by Hsp70. Heat shock protein-40 (Hsp40) heat shock proteins are essential for the in vivo function of Hsp70s by stimulating the ATPase activity of these proteins and facilitating transfer of substrates. The Type III class of Hsp40 proteins have not been well characterised due to their poor levels of conservation at the primary sequence level. This is due to the fact that Type III Hsp40s only contain a J-domain and a poorly conserved C-terminal region. The newly identified Type IV class of Hsp40s, contain an abrogated HPD tripeptide motif in the J-domain and have also not been extensively studied. Trypanosoma brucei (T. brucei) is a unicellular flagellated protozoan parasite. It is the causative agent of Human African Trypansomiasis (HAT) which results in thousands of deaths and devastating agricultural losses in many parts of Africa. T. brucei undergoes a complex lifecycle that is characterised by the transition from an insect vector to a mammalian host in markedly different conditions of temperature, pH, nutrient availability and respiratory requirements. It has been proposed that molecular chaperones may enhance the survival of these parasites due to their cytoprotective effect in combating cellular stress. Due to the fact that T. brucei infection is invariably fatal if left untreated, and that no novel treatment regimens have been developed recently, the identification of potential novel drug targets among proteins essential to the parasite’s survival in the host organism is an attractive aspect of T. brucei research. Because Type III Hsp40s are poorly conserved with respect to Hsp40s found in the human host, the identification of any of these proteins found to be essential to T. brucei survival in humans could potentially make attractive novel drug targets. An in depth in silico investigation into the Type III Hsp40 complement as well as partner Hsp70 proteins in T.brucei was performed. T. brucei possesses 65 Hsp40 proteins, of which 47 were classed as Type III and 6 of which were identified as being putative Type IV Hsp40s. A small but significant number (5) of Type III TbHsp40s contained tetratricopeptide (TPR) domains in addition to the J-domain. The J-domains of the Type III TbHsp40 complement were found to be conserved with respect to those of canonical Hsp40 proteins, although the mutation of certain residues that play a key role in Hsp40-Hsp70 interaction was noted. Potential partnerships of these proteins in the parasite was also investigated. The coding regions of three previously uncharacterised TbHsp40s were successfully amplified from T. brucei TREU927 genomic DNA and cloned into an expression vector. Tbj1, a Tcj1 ortholog, was selected for further study and successfully expressed and biochemically characterised. Tbj1 expressed in E. coli was found to be insoluble, but large amounts were recovered with the aid of a denaturing purification followed by refolding elution strategies, and the bulk of the protein recovered was in compact monomeric form as determined by size-exclusion chromatography fast protein liquid chromatography (SEC-FPLC). The addition of Tbj1 to a thermally aggregated substrate resulted in increased levels of aggregation, although Tbj1 was able to assist two Hsp70 proteins in the suppression of aggregation. Tbj1 proved unable to stimulate the ATPase activity of these same Hsp70s, and could not rescue temperature sensitive cells when replacing E.coli DnaJ and CbpA. It was concluded that Tbj1 does not possess independent chaperone activity, but could display Hsp40 co-chaperone properties under certain circumstances. This could allude to a specialised function in the T. brucei parasite. The lack of human orthologues to Tbj1 could result in the attractiveness of this protein as a novel drug target
Characterisation of Trypanosomal Type III and Type IV Hsp40 proteins
The heat shock protein-70 (Hsp70) family of molecular chaperones are ubiquitous highly conserved proteins that are critical for the viability of cellular homeostasis. The ATPase activity of Hsp70 proteins is critical to their function as the affinity of a given Hsp70 for non-native substrate is modulated by ATP binding and hydrolysis. When bound to ATP, Hsp70s possess a low affinity for a given substrate protein, while the hydrolysis of ATP to ADP causes a conformational change that results in a high affinity for substrate proteins. The basal ATPase activity of Hsp70s is too low to facilitate their function in vivo, and co-chaperones are essential to modulate the efficient protein folding by Hsp70. Heat shock protein-40 (Hsp40) heat shock proteins are essential for the in vivo function of Hsp70s by stimulating the ATPase activity of these proteins and facilitating transfer of substrates. The Type III class of Hsp40 proteins have not been well characterised due to their poor levels of conservation at the primary sequence level. This is due to the fact that Type III Hsp40s only contain a J-domain and a poorly conserved C-terminal region. The newly identified Type IV class of Hsp40s, contain an abrogated HPD tripeptide motif in the J-domain and have also not been extensively studied. Trypanosoma brucei (T. brucei) is a unicellular flagellated protozoan parasite. It is the causative agent of Human African Trypansomiasis (HAT) which results in thousands of deaths and devastating agricultural losses in many parts of Africa. T. brucei undergoes a complex lifecycle that is characterised by the transition from an insect vector to a mammalian host in markedly different conditions of temperature, pH, nutrient availability and respiratory requirements. It has been proposed that molecular chaperones may enhance the survival of these parasites due to their cytoprotective effect in combating cellular stress. Due to the fact that T. brucei infection is invariably fatal if left untreated, and that no novel treatment regimens have been developed recently, the identification of potential novel drug targets among proteins essential to the parasite’s survival in the host organism is an attractive aspect of T. brucei research. Because Type III Hsp40s are poorly conserved with respect to Hsp40s found in the human host, the identification of any of these proteins found to be essential to T. brucei survival in humans could potentially make attractive novel drug targets. An in depth in silico investigation into the Type III Hsp40 complement as well as partner Hsp70 proteins in T.brucei was performed. T. brucei possesses 65 Hsp40 proteins, of which 47 were classed as Type III and 6 of which were identified as being putative Type IV Hsp40s. A small but significant number (5) of Type III TbHsp40s contained tetratricopeptide (TPR) domains in addition to the J-domain. The J-domains of the Type III TbHsp40 complement were found to be conserved with respect to those of canonical Hsp40 proteins, although the mutation of certain residues that play a key role in Hsp40-Hsp70 interaction was noted. Potential partnerships of these proteins in the parasite was also investigated. The coding regions of three previously uncharacterised TbHsp40s were successfully amplified from T. brucei TREU927 genomic DNA and cloned into an expression vector. Tbj1, a Tcj1 ortholog, was selected for further study and successfully expressed and biochemically characterised. Tbj1 expressed in E. coli was found to be insoluble, but large amounts were recovered with the aid of a denaturing purification followed by refolding elution strategies, and the bulk of the protein recovered was in compact monomeric form as determined by size-exclusion chromatography fast protein liquid chromatography (SEC-FPLC). The addition of Tbj1 to a thermally aggregated substrate resulted in increased levels of aggregation, although Tbj1 was able to assist two Hsp70 proteins in the suppression of aggregation. Tbj1 proved unable to stimulate the ATPase activity of these same Hsp70s, and could not rescue temperature sensitive cells when replacing E.coli DnaJ and CbpA. It was concluded that Tbj1 does not possess independent chaperone activity, but could display Hsp40 co-chaperone properties under certain circumstances. This could allude to a specialised function in the T. brucei parasite. The lack of human orthologues to Tbj1 could result in the attractiveness of this protein as a novel drug target
The role of O-GlcNAc and acetylation on the pathogenesis of Alzheimers disease
학위논문 (박사)-- 서울대학교 대학원 : 의과대학 의과학과, 2018. 8. 묵인희.알츠하이머병에서 주요 원인 물질인 베타-아밀로이드와 tau 단백질에 의한 세포 독성으로 당 대사의 저하로 인한 O-GlcNAcylation의 변화와 HDAC6의 과활성화로 인한 acetylation의 변화가 알려져 있다. 하지만, O-GlcNAcylation이나 acetylation의 변화가 관여하는 구체적인 알츠하이머병의 병인 기전은 많이 연구되어 있지 않다. 따라서, 본 연구에서는 O-GlcNAcylation이나 acetylation의 변화가 알츠하이머병의 병인 기전에 미치는 영향을 알아보고자 하였다.
먼저, 알츠하이머병에서 변화된 O-GlcNAcylation이 기여하는 병인 기전 연구에서는, 알츠하이머병 동물 모델과 베타-아밀로이드를 처리한 세포에서 c-Fos의 O-GlcNAcylation (S56, S57)이 증가함을 밝혔으며, 그 결과 c-Fos의 안정화와 전사 활성의 증가로 세포 사멸을 유도하는 Bim의 발현이 증가되어 세포 사멸이 촉진됨을 확인하였다.
다음으로, acetylation 연구에서는 HDAC6 억제제를 통한 acetylation 회복에 의해 나타나는 알츠하이머병 회복 기전을 규명하였다. 알츠하이머병 동물 모델이나 세포에 HDAC6억제제를 처리하였을 때, HDAC6의 기질인 Prx1의 acetylation (K197)이 증가하여 베타-아밀로이드에 의해 유도된 산화 스트레스와 과도한 Ca2+을 회복시키고, 그 결과 미토콘드리아의 축삭 수송도 회복시킴을 확인하였다. 또, tau와 Hsc70, Hsp70의 acetylation이 증가하여 서로 간의 상호 작용 증가를 통해 tau의 프로테아좀으로의 분해가 촉진됨을 밝혔고, 그 결과 응집된 tau를 포함한 전체적인 tau의 양이 감소하여, 손상된 시냅스와 기억력이 회복됨을 확인하였다.
이러한 결과를 통해서 알츠하이머병에서 변화된 O-GlcNAcylation, acetylation과 같은 단백질의 번역 후 수식화가 알츠하이머병의 병인 기전에 기여함을 알 수 있었고, 변화된 번역 후 수식화를 회복시켜 줌으로써 알츠하이머병의 병적 증상을 회복시킬 수 있음을 확인할 수 있었다. 따라서, 단백질의 번역 후 수식화를 조절하는 것은 알츠하이머병의 중요한 치료 표적이 될 수 있을 것으로 기대된다.초록 i
도표 및 약어 목록 iii
목차 v
표 및 그림 목록 vi
서론 1
실험재료 및 방법 26
결과 48
고찰 120
결론 140
참고문헌 142
초록 (영문) 164Docto
An analysis of the NET1 proteins: a group of novel plant actin-binding proteins
The NET protein superfamily is a recently discovered family of novel, plant-specific actin binding proteins. The identification of this family represents a significant discovery as the plant cytoskeleton is not identical to the animal cytoskeleton and plant cells show plant specific processes and subcellular structures which rely on actin. There is a need for plant specific proteins which are capable of modelling actin within plant cells.
The NET family comprises thirteen proteins in Arabidopsis thaliana, which share the NET actin binding domain (found at the N-terminal end of each protein). Based on the C-terminal domains of the proteins, the family can be separated into four groups, each with a particular localisation. The localisations of these proteins are frequently within plant specific cell types, such as pollen tubes, guard cells or roots, or to plant specific cell structures such as plasmodesmata. It is thought that these proteins may be involved in modelling the actin cytoskeleton at junctions between actin and membranes (either cell membranes or membrane bound structures such as the endoplasmic reticulum).
The NET1 proteins are a group of four proteins, each consisting of an N-terminal actin binding domain and C-terminal coiled-coil domains. NET1a was the first protein to be discovered in a high-throughput screen of plant proteins for novel localisations carried out by Karl Oparka, where it was shown to bind to filaments. Work by Calcutt (Calcutt 2009) showed the filaments to be the actin cytoskeleton.
The aim of this thesis has been to complete the characterisation of all Group 1 NET proteins, building on the analysis of NET1a by Calcutt (Calcutt 2009) and to investigate the possible functions of the NET1 subfamily proteins. All four proteins have been shown to be capable of actin binding and to be expressed in, and locate to structures within, the roots of A. thaliana. NET1a has been linked to plasmodesmata, and the combined absence of NET1a and NET1b in the plant results in a cumulative, long root phenotype. Theories to explain this phenotype are suggested here, although the validation and testing of these will form the basis of future work
Ex vivo μελέτη των κυτταρικών αποκρίσεων του ανθρώπινου πολφού σε συγκολλητικούς παράγοντες κατά τα πρώιμα στάδια της διεργασίας επούλωσης
Σκοπός:
Ο σκοπός της παρούσας ex vivo μελέτης ήταν η εξέταση των πρώιμων κυτταρικών αποκρίσεων του πολφού στην άμεση κάλυψή του με τρία συγκολλητικά συστήματα συνθέτων ρητινών (Prime&Bond NT, Clearfil SE Bond και Clearfil S3 Bond) και το Dycal. Οι παράμετροι που εξετάστηκαν περιλάμβαναν την ζωτικότητα και την ιστική ακεραιότητα του πολφού, την απόπτωση των κυττάρων του καθώς και την έκφραση της Βιμεντίνης, του ERdj5 και του TGFβ2.
Υλικά και Μέθοδος:
Τα συγκολλητικά συστήματα, το Dycal και το ρυθμιστικό διάλυμα (DPBS) εφαρμόστηκαν άμεσα σε τομές πολφού ανθρώπινων δοντιών διατηρημένων σε θρεπτικό μέσο κυτταροκαλλιέργειας για 4 μέρες. Η εκτίμηση της ζωτικότητας του ιστού κατά τη διάρκεια της παραμονής του σε καλλιέργεια έγινε με την εφαρμογή της χρώσης ΜΤΤ. Μετά το πέρας των τεσσάρων ημερών, η ακεραιότητα του ιστού εκτιμήθηκε με τη χρώση αιματοξυλίνης-ηωσίνης, την ανοσοϊστοχημική χρώση και την ανοσοστύπωση κατά Western για τη Βιμεντίνη, καθώς και με τη μέθοδο TUNEL για τον εντοπισμό των αποπτωτικών κυττάρων. Η εκτίμηση του στρες του Ενδοπλασματικού Δικτύου και της ιστικής ανάπλασης πραγματοποιήθηκε με την ανοσοϊστοχημική χρώση και την ανοσοστύπωση κατά Western για την πρωτεΐνη-δείκτη στρες του ενδοπλασματικού δικτύου ERdj5 και τον αυξητικό παράγοντα TGFβ2, αντίστοιχα.
Αποτελέσματα:
Τα αποτελέσματα της μελέτης έδειξαν ότι οι πρώιμες αποκρίσεις του πολφού στην άμεση κάλυψή του με τους εξεταζόμενους συγκολλητικούς παράγοντες και το Dycal διαφέρουν σημαντικά. Σημαντική μείωση της ζωτικότητας των κυττάρων και διατάραξη της ακεραιότητας του ιστού παρατηρήθηκε στις ομάδες όπου εφαρμόστηκαν τα συγκολλητικά συστήματα. Λιγότερο σημαντική επίδραση είχε η εφαρμογή του Dycal. Εκτεταμένη απόπτωση προκάλεσαν το Clearfil SE και το Prime&Bond. Το Dycal προκάλεσε περιορισμένη απόπτωση στην περιοχή της εφαρμογής του. Όλα τα συγκολλητικά συστήματα μείωσαν τα επίπεδα της Βιμεντίνης και του TGFβ2 στην περιοχή της εφαρμογής. Τα συνολικά επίπεδα της Βιμεντίνης στον πολφό μειώθηκαν επίσης. Μειωμένα επίπεδα της ERdj5 παρατηρήθηκαν στην περιοχή εφαρμογής του Prime&Bond ενώ αυξημένα επίπεδα της ERdj5 παρατηρήθηκαν στις παρακείμενες περιοχές προς την περιοχή της εφαρμογής του Prime&Bond και του Dycal.
Συμπεράσματα:
Τα παραπάνω αποτελέσματα υποδεικνύουν την ανάγκη περαιτέρω διευκρίνησης των τυχόν αρνητικών επιπτώσεων των συγκολλητικών συστημάτων στον οδοντικό πολφό, με τη διενέργεια συγκριτικών μελετών των κυτταρικών αποκρίσεων του πολφού στα χρησιμοποιούμενα συστήματα. Εx vivo μοντέλα μελέτης, όπως αυτό που χρησιμοποιήθηκε στην παρούσα διατριβή, θα μπορούσαν να εξυπηρετήσουν αυτόν το στόχο.Early responses of human pulp to Prime&Bond / phosphoric acid, Clearfil SE Bond, Clearfil S3 Bond and Dycal were investigated ex vivo.
The three adhesives, Dycal or buffer (DPBS) were applied directly onto the pulp of human teeth slices that were placed in culture for 4 days. Cell viability was monitored by the MTT assay during the culture period. After 4 days, tissue integrity was examined by hematoxylin-eosin staining, Vimentin immunostaining and Western blotting, and TUNEL assay for apoptotic cells. Endoplasmic reticulum stress and tissue repair were assessed through the levels of ERdj5 and TGFβ2 determined by immunohistochemistry and Western blotting.
Profound reduction of cell viability and tissue integrity was observed in adhesives’-treated groups, while the impact of Dycal was less harmful. Extended apoptosis was caused by the Clearfil SE and Prime&Bond, while in Dycal group apoptosis was limited to the application area. All adhesives reduced Vimentin and TGFβ2 levels at the application site. Total Vimentin levels were reduced as well. Increased ERdj5 levels were detected in adjacent to the treatment areas of Prime&Bond- and Dycal-treated groups.
These results provide evidence that the early pulp responses to different capping materials may vary significantly and underline the need for further mechanistic studies in relevant ex vivo systems