Marijanović Inga, Kronenberg Mark S, Erceg Ivkosić Ivana, Lichtler Alexander C
Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut, USA.
Coll Antropol. 2009 Sep;33(3):919-24.
We analyzed proliferation and differentiation of calvarial osteoblasts derived from Msx2 deficient in comparison with wild type mice. Calvarial osteoblast cultures from five to eight days old Msx2 deficient, heterozygous and wild type mice were studied for difference in proliferation and differentiation. Proliferation rate was assessed by counting cell number, BrdU and Calcein AM labeling. Differentiation was assessed by Von Kossa and alkaline phosphatase staining, northern blot hybridization with bone differentiation markers, infection of cell cultures with retrovirus expressing GFP under the control of type I collagen promoter fragment. At day six, cell number in cell culture derived from Msx2 deficient mice was 20% lower then in culture from wild type mice. There were 16.8% BrdU labeled cells in cell culture from Msx2 deficient mice, 20.9% in culture from heterozygous mice and 21.6% in culture from wild type mice. Cell cultures from Msx2 deficient mice showed lower intensity of fluorescence when marked with Calcein AM then cultures from wild type mice. Von Kossa staining showed increased mineralization and northern blot analysis showed increased levels of bone differentiation markers in cell cultures derived from Msx2 deficient mice. GFP came on earlier in Msx2 deficient cultures after infection with Col2.3 GFP retrovirus. We conclude that calvarial osteoblasts derived from Msx2 deficient mice have a lower rate of proliferation and demonstrate increased osteoblastic differentiation when compared to osteoblasts derived from wild type mice.
我们分析了与野生型小鼠相比，Msx2基因缺陷型小鼠颅骨成骨细胞的增殖和分化情况。研究了来自5至8日龄Msx2基因缺陷型、杂合子型和野生型小鼠的颅骨成骨细胞培养物在增殖和分化方面的差异。通过计数细胞数量、BrdU和钙黄绿素AM标记来评估增殖率。通过冯·科萨染色和碱性磷酸酶染色、与骨分化标志物进行Northern印迹杂交、用在I型胶原启动子片段控制下表达绿色荧光蛋白的逆转录病毒感染细胞培养物来评估分化情况。在第6天，来自Msx2基因缺陷型小鼠的细胞培养物中的细胞数量比来自野生型小鼠的培养物低20%。来自Msx2基因缺陷型小鼠的细胞培养物中有16.8%的细胞被BrdU标记，来自杂合子型小鼠的培养物中为20.9%，来自野生型小鼠的培养物中为21.6%。用钙黄绿素AM标记时，来自Msx2基因缺陷型小鼠的细胞培养物显示出比来自野生型小鼠的培养物更低的荧光强度。冯·科萨染色显示矿化增加，Northern印迹分析显示来自Msx2基因缺陷型小鼠的细胞培养物中骨分化标志物水平增加。用Col2.3 GFP逆转录病毒感染后，Msx2基因缺陷型培养物中绿色荧光蛋白更早表达。我们得出结论，与来自野生型小鼠的成骨细胞相比，来自Msx2基因缺陷型小鼠的颅骨成骨细胞增殖率较低，且成骨细胞分化增加。
