Zero Order


An update on MMP production

Posted in Uncategorized by Amy Ross on January 16, 2011

Soooooo..in a comment to my last entry, I remarked that not only was it possible my cells weren’t producing enough active MMP-2 to produce a measurable release of peptide in the hydrogels, I thought it was likely. It’s a hypothesis I’ve been pursuing for about a month. As we’ve talked about before, cell lines in culture don’t always behave the same way as their primary cell counterparts in vivo. They’re different genetically, they don’t produce MMP while living on tissue culture plastic…there could be any number of reasons.

I started looking at papers on MMP-2 expression, and how other groups boosted theirs. One option is to use chemicals to convert the inactive form of the enzyme to the active form. I found one paper that used plates coated in extracellular matrix proteins boosted their MMP-2 presence.

This was a fairly easy hypothesis to test. I ran two experiments. In one, I did an in-vitro peptide release study, and saved the media at each time point.  In the second, I collected media after 48 hours of exposures to my cells, and exposed hydrogels to this media (hereafter referred to as conditioned media). I ran gel zymography on the samples from the in vitro study and the conditioned media study.

The results? Unsupportive of my hypothesis. The gel showed plenty of active MMP-2 production by the 48 hour mark in the in vitro study and in the conditioned media study. However, there was no significant difference in peptide release compared to the controls (in both cases, hydrogels in media only).

Sigh.

Next hypothesis: the MMP-2 may be there, but is it active? Testing this at the moment with mixed results. Hopefully I”ll have more to talk about later!

In other news, my wonderful husband gave me an iPad for Christmas. I’m thinking of downloading the Keynote app and doing my group meeting presentations from the iPad. One of our rotation students from last semester did some nice work with Keynote. I think incorporating some more of the sophisticated animations could be useful without being obnoxious.

 

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