Veränderung des Zell-Phänotyps durch Morbus Dupuytren – eine in-vitro-Analyse^[*]

 

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Zusammenfassung

Einleitung:

Goldstandard in der Therapie des fortgeschrittenen Morbus Dupuytren ist nach wie vor die partielle Aponeurektomie mit allen bekannten Nebenwirkungen. Mit dem Ziel, neue Therapien zu entwickeln, haben wir Zellen aus gesunder Palmaraponeurose (Kon) isoliert, molekularbiologisch charakterisiert und mit Zelliso­laten von Patienten mit Erstmanifestation von Morbus Dupuytren (PrimDup) sowie von Patienten mit Morbus-Dupuytren-Rezidiv (RezDup) verglichen. Da Zellen der Palmaraponeurose von Morbus-Dupuytren-Patienten Merkmale von Stammzellen innehaben, wurde neben der Zellmorphologie und dem Integrinprofil der Zellen insbesondere das Wachstumsverhalten und der Stammzellcharakter untersucht.

Material und Methoden:

Aus 15 Dupuytren-Präparaten von partiellen Aponeurektomien wurden je 3 Spender (RezDup, PrimDup) ohne extrinsische Risikofaktoren selektiert. Gesunde Spender dienten als Kontrolle (Kon). Die Zellen wurden isoliert und expandiert. Wachstumskurven der 3 Zelltypen wurden erstellt. Die Zellen wurden nach etablierten Protokollen osteogen und adipogen differenziert. Eine semiquantita­tive PCR Analyse der Integrine α2, β3, β5 und Fibronektin wurde durchgeführt.

Ergebnisse:

PrimDup-Zellen proliferierten signifikant schneller als Kon-Zellen, die wiederum schneller proliferierten als RezDup-Zellen. PrimDup- und Kon-Zellen gelangten nach ca. 40 Tagen in Seneszenz, während RezDup-Zellen über 100 Tage in Zellkultur proliferierten. RezDup- und PrimDup-Zellen differenzierten am stärksten adipogen und osteogen. Demgegenüber differenzierten Kon-Zellen nur mäßig. Die PCR-Analyse der Integrine zeigte, dass die Fibronektin-bindenden Integrine β3 und β5 in PrimDup- und RezDup-Zellen im Vergleich zu gesunden Zellen der Palmaraponeurose vermehrt exprimiert wurden.

Schlussfolgerungen:

PrimDup-Zellen wachsen schneller als die anderen Zelltypen, was nahelegt, dass hier eine Störung der Wachstumsregulation vorliegt. Da RezDup-Zellen innerhalb von 100 Tagen nicht in Seneszenz gelangen, muss angenommen werden, dass diese Zellen Defekte in Seneszenz-kontrollierenden Mechanismen haben. Sowohl RezDup- als auch PrimDup-Zellen zeigten eine bessere adipogene und osteogene Differenzierbarkeit als die gesunden Kontrollen, was darauf hinweist, dass Morbus Dupuytren eine weniger differenzierte Vorstufe von Fibroblasten induziert. Dass die Fibronektin-bindenden Integrine β3 und β5 bei Morbus Dupuytren erhöht sind zeigt, dass Zell-Matrix-Interaktionen verändert sind. Hier könnte ein Ansatzpunkt für zukünftige Therapieansätze liegen.

Abstract

Introduction:

Despite its potential complications, partial aponeurectomy still is the mainstay of treatment whenever it comes to significant contracture in Dupuytren’s disease. With the goal in mind to identify new therapeutic strategies we isolated and characterised cells from healthy palmar aponeurosis (Kon) and compared them to cells isolated from palmar aponeurosis of patients with a primary manifestation of Dupuytren’s disease (PrimDup) as well as from patients with recurrent Dupuytren’s disease (RezDup). As cells from palmar aponeurosis from patients with Dupuytren’s disease share characteristics with stem cells, such as the ability to differentiate into other cell types, we analysed the stemness, morphology and integrin receptor profiles of the cells.

Materials and Methods:

A total of 15 Dupuytren samples were collected from regular partial aponeurectomy procedures. From these, 3 donors without extrinsic risk factors were selected per group (RezDup, PrimDup). Cells were isolated and expanded under standard cell culture conditions. Cells from healthy patients served as control (Kon). Growth curves were produced. Cells were subjected to osteogenic and adipogenic differentiation using standard protocols. Semiquantitative PCR analysis of the integrins α2, β3, β5 and fibronectin was performed.

Results:

PrimDup cells proliferated significantly faster than control cells, which in turn proliferated faster than RezDup cells. Both PrimDup and control cells went into senescence after approximately 40 days whereas RezDup cells proliferated over the entire period of 100 days. Osteogenic and adipogenic differentiation was best in cells derived from Dupuytren patients while Kon cells differentiated poorly. PCR analysis revealed that fibronectin-binding integrins β3 and β5 are upregulated in Dupuytren’s disease.

Conclusions:

PrimDup cells grow faster than the other cell types suggesting that their growth regulation may be altered. The fact that RezDup cells do not reach senescence over 100 days in culture indicates that senescence regulating factors may be altered. As cells from Dupuytren patients differentiate better along the osteogenic and adipogenic lineages, they probably possess a higher level of stemness. Their modified integrin profile may be a key to future therapies.

^*  Nach einem Vortrag beim Jahreskongress der Deutschen Gesellschaft für Handchirurgie (DGH) vom 6.10.2011–8.10.2011 in Bonn

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